Crystalline l-arginine salt of (r)-2-(7-(4-cyclopentyl-3- (trifluoromethyl)benzyloxy)-1,2,3, 4-tetrahydrocyclo-penta [b]indol-3-yl)acetic acid(compound1) for use in sipi receptor-associated disorders

ABSTRACT

The present invention relates to, inter alia, a novel crystalline free-plate habit or morphology, processes for preparing the crystalline free-plate habit, and uses of the crystalline free-plate habit of the L-arginine salt of (R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)acetic acid (Compound 1) in the treatment of S1P 1  receptor-associated disorders, for example, diseases and disorders mediated by lymphocytes, transplant rejection, autoimmune diseases and disorders, inflammatory diseases and disorders (e.g., acute and chronic inflammatory conditions), cancer, and conditions characterized by an underlying defect in the vascular integrity or that are associated with angiogenesis such as may be pathologic (e.g., as may occur in inflammation, tumor development, and atherosclerosis).

FIELD OF THE INVENTION

The present invention relates to, inter alia, a novel crystallinefree-plate habit or morphology, processes for preparing the crystallinefree-plate habit, and uses of the crystalline free-plate habit of theL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid (Compound 1) in the treatment of S1P₁ receptor-associateddisorders, for example, diseases and disorders mediated by lymphocytes,transplant rejection, autoimmune diseases and disorders, inflammatorydiseases and disorders (e.g., acute and chronic inflammatoryconditions), cancer, and conditions characterized by an underlyingdefect in the vascular integrity or that are associated withpathological angiogenesis (e.g., as may occur in inflammation, tumordevelopment, and atherosclerosis).

BACKGROUND OF THE INVENTION

The sphingosine-1-phosphate (S1P) receptors 1-5 constitute a family of Gprotein-coupled receptors with a seven-transmembrane domain. Thesereceptors, referred to as S1P₁ to S1P₅ (formerly termed endothelialdifferentiation gene (EDG) receptor-1, -5, -3, -6, and -8, respectively;Chun et. al., Pharmacological Reviews, 54:265-269, 2002), are activatedvia binding by sphingosine-1-phosphate, which is produced by thesphingosine kinase-catalyzed phosphorylation of sphingosine. S1P₁, S1P₄,and S1P₅ receptors activate Gi but not Gq, whereas S1P₂ and S1P₃receptors activate both Gi and Gq. The S1P₃ receptor, but not the S1P₁receptor, responds to an agonist with an increase in intracellularcalcium.

The compound(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid (Compound 1) is a potent (EC₅₀ cAMP, 0.093 nM (human)) andselective (EC₅₀ β-arrestin, 6.10 nM (S1P₁), >10,000 nM (S1P₂), >10,000nM, (S1P₃), 147 nM (S1P₄), and 24.4 nM (S1P₅)), orally availableinvestigational drug candidate for the S1P₁ receptor.

In preclinical studies, Compound 1 showed calculated lymphocyte loweringIC₅₀ values in four different species: 0.101 μM (mouse), 0.051 μM (rat),0.058 μM (dog), and 0.098 μM (monkey). Notably, the calculatedlymphocyte lowering IC₅₀ values reflect total plasma concentrationwherein Compound 1 is highly protein bound (97.8% human, 98.0% rat).Compound 1 was shown to be efficacious in the murine experimentalautoimmune encephalomyelitis (EAE) model that mimics multiple sclerosis.Prophylactically, Compound 1 prevented the onset and severity of diseaserelative to vehicle up to day 25, at which time dosing was discontinued.All treatment arms went on to develop severe disease. Therapeuticadministration of Compound 1 was also examined. Treatment began at day18, by which time all animals had developed severe disease. Compound 1was administered from day 18 to day 37 and showed to reverse the diseaserelative to vehicle and was similar to the efficacy observed withfingolimod (i.e., GILENYA® was approved in September 2010 for thetreatment of patients with relapsing forms of multiple sclerosis).Similarly, Compound 1 was efficacious in a collagen induced arthritis(CIA) model. Prophylactic oral administration in female Lewis ratsresulted in a significant reduction in ankle diameters on day 17following a daily oral dose and was similar to that observed in ratstreated with fingolimod or methotrexate. Improvement in histologicalparameters in the knees and ankles of CIA rats was also observed,suggesting that inhibiting lymphocyte entry into arthritic joints withCompound 1 treatment suppresses CIA in rodents. Additional details canbe found in the following, PCT application, serial numberPCT/US2009/004265, filed 22 Jul. 2009 (International Publication NumberWO2010/011316); PCT application, serial number PCT/US2011/000153, filed27 Jan. 2011 (International Publication Number WO2011/094008); andBuzard: D. J., et. al., ACSMed. Chem. Lett. 2014, 5, 1313-1317; eachhereby incorporated by reference in its entirety.

The L-arginine salt of Compound 1 was selected for clinical evaluation.A randomized, double-blind, placebo-controlled Phase 1b clinical trialwas conducted to evaluate the safety, tolerability, pharmacodynamics,and pharmacokinetics of multiple-ascending doses of the L-arginine saltof Compound 1 in five different dosing cohorts. A total of 50 healthyvolunteers received the L-arginine salt of Compound 1 and 10 healthyvolunteers received placebo for 21 days. In the Phase 1b clinical trial,the administration of the L-arginine salt of Compound 1 demonstrated adose-dependent effect on lymphocyte count lowering in blood, with a meandecrease from baseline of up to 69%. Lymphocyte counts, on average,recovered to baseline within one week of the conclusion of dosing.

In view of the growing demand for compounds useful in the treatment ofS1P₁ receptor-associated disorders, the L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid (Compound 1) has emerged as an important new compound.

Accordingly, new and efficient methods are needed for the preparation ofthe L-arginine salt of Compound 1 and crystalline morphologies relatedthereto. Several improvements have now been discovered in this regard.These improvements are described herein.

Citation of any reference throughout this application is not to beconstrued as an admission that such reference is prior art to thepresent application.

SUMMARY OF THE INVENTION

The present invention relates to, inter alia, a novel crystallinefree-plate habit or morphology, processes for preparing the crystallinefree-plate habit, and uses of the crystalline free-plate habit of theL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid (Compound 1) in the treatment of S1P₁ receptor-associateddisorders, such as, those described herein.

Discovery of the novel crystalline plate habit, referred to as“free-plate habit” herein, led to certain advantageous properties, suchas, improved in-process filtrations, formulation stability and/orstability to degradation, higher degree of crystallinity, and lowerdegree of hygroscopicity, see Example 8 for details.

Certain processes for the preparation of the L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid have been previously described; see WO2010/011316 andWO2011/094008.

One aspect of the present invention relates to methods for preparing acrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, the method comprising the steps of:

-   -   a) hydrolyzing a compound of Formula (IIa):

wherein R³ is C₁-C₆ alkyl; in the presence of a hydrolyzing mixturecomprising a lipase and a hydrolyzing-step solvent to form(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid;

b) adding L-arginine to a salt-forming mixture comprising(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, a water-miscible anti-solvent, and H₂O to form a first mixturecomprising L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid;

c) heating the first mixture to a first heating temperature;

d) adding a first additional amount of the water-miscible anti-solventto the first mixture to form a suspension; and

e) cooling the suspension to form the crystalline free-plate habit ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.

One aspect of the present invention relates to methods for preparing acrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, said method comprising the steps of:

a) hydrolyzing a compound of Formula (IIa):

wherein R³ is C₁-C₆ alkyl; in the presence of a hydrolyzing mixturecomprising a lipase and a hydrolyzing-step solvent to form(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid;

b) forming a first mixture comprising L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, a water-miscible anti-solvent, and H₂O;

c) heating the first mixture to a first heating temperature to form asecond mixture;

d) adding a first additional amount of said water-miscible anti-solventto said second mixture while maintaining said first heating temperatureto form a suspension;

e) cooling said suspension to a first cooling temperature and thereafterheating to a second heating temperature;

f) cycling Step e) optionally one or more times, wherein said firstcooling temperature at each cycle may be the same or different and saidsecond heating temperature at each cycle may be the same or different;and

g) cooling said suspension to a final cooling temperature to form saidcrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.

One aspect of the present invention relates to methods for preparing acrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, the method comprising the steps of:

a) adding L-arginine to a salt-forming mixture comprising(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, a water-miscible anti-solvent, and H₂O to form a first mixturecomprising L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid;

b) heating the first mixture to a first temperature;

c) adding a first additional amount of the water-miscible anti-solventto the first mixture to form a suspension; and

d) cooling the suspension to form the crystalline free-plate habit ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.

One aspect of the present invention relates to methods for preparing acrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, the method comprising the steps of:

a) heating a first mixture comprising L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, a water-miscible anti-solvent, and water to a first heatingtemperature to form a second mixture;

b) cooling the second mixture to a first cooling temperature followed byadding a first additional amount of the water-miscible anti-solvent tothe second mixture while maintaining the first cooling temperature andthereafter heating to a second heating temperature to form a suspension;

c) cycling Step b) optionally once or twice; and

d) cooling the suspension to a second cooling temperature to form thecrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.

One aspect of the present invention relates to methods for preparing acrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, said method comprising the steps of:

a) forming a first mixture comprising L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, a water-miscible anti-solvent, and H₂O;

b) heating the first mixture to a first heating temperature to form asecond mixture;

c) adding a first additional amount of said water-miscible anti-solventto said second mixture while maintaining said first heating temperatureto form a suspension;

d) cooling the suspension to a first cooling temperature and thereafterheating to a second heating temperature;

e) cycling Step d) optionally one or more times, wherein the firstcooling temperature at each cycle may be the same or different and thesecond heating temperature at each cycle may be the same or different;and

f) cooling said suspension to a final cooling temperature to form saidcrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.

One aspect of the present invention relates to methods for preparing acrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid further comprising the step of formulating the crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid to form a pharmaceutical composition.

One aspect of the present invention relates to methods for preparing acrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid further comprising the step of admixing the crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid with a pharmaceutical excipient to form a pharmaceuticalcomposition.

One aspect of the present invention relates to pharmaceuticalcompositions prepared according to any of the methods described herein.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid prepared according to any of the methods described herein.

One aspect of the present invention relates to compositions comprising acrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid prepared according to any of the methods described herein.

One aspect of the present invention relates to pharmaceuticalcompositions comprising a crystalline free-plate habit of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid prepared according to any of the methods described herein and apharmaceutical excipient.

One aspect of the present invention relates to pharmaceuticalcompositions comprising a crystalline free-plate habit of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid (Compound 1) in an amount equivalent to a therapeutically effectiveamount of Compound 1 of the crystalline free-plate habit, wherein thepharmaceutical composition further comprises a diluent, a disintegrant,and a lubricant.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid having a powder X-ray diffraction pattern comprising peaks, interms of 2θ, at 8.2°±0.2°, 16.40°±0.2°, and 20.5°±0.2°.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid having a differential scanning calorimetry trace comprising anendotherm with an extrapolated onset temperature of 205.0° C. to 208.5°C. when scanned at 10° C. per minute.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid having a dynamic moisture sorption (DMS) profile with an adsorptionphase from 30% RH to 90% RH wherein said crystalline free-plate habitgains about 0.3% weight or less at 90% RH.

One aspect of the present invention relates to compositions comprising acrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid as described herein.

One aspect of the present invention relates to pharmaceuticalcompositions comprising a crystalline free-plate habit of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid as described herein and a pharmaceutical excipient.

One aspect of the present invention relates to methods for treating anS1P₁ receptor-associated disorder in an individual comprisingadministering to the individual in need thereof a therapeuticallyeffective amount of a crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein, a composition as described herein, or apharmaceutical composition as described herein.

One aspect of the present invention relates to uses of a crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein in the manufacture of a medicament for thetreatment of an S1P₁ receptor-associated disorder.

One aspect of the present invention relates to uses of a compositioncomprising a crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein and an excipient in the manufacture of amedicament for administration in the treatment of an S1P₁receptor-associated disorder.

One aspect of the present invention relates to uses of a compositioncomprising a crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein and an excipient for preparation a medicamentfor administration in the treatment of an S1P₁ receptor-associateddisorder.

One aspect of the present invention relates to uses of a compositioncomprising a crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein and an excipient for compounding a medicamentfor administration in the treatment of an S1P₁ receptor-associateddisorder.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein for use in a method for the treatment of thehuman or animal body by therapy.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein for use in a method for the treatment of anS1P₁ receptor-associated disorder.

In some embodiments, the S1P₁ receptor-associated disorder is selectedfrom: primary biliary cirrhosis, psoriasis, psoriatic arthritis,rheumatoid arthritis, Crohn's disease, multiple sclerosis, inflammatorybowel disease, systemic lupus erythematosus, and ulcerative colitis.

These and other aspects of the invention disclosed herein will be setforth in greater detail as the patent disclosure proceeds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a micrograph of spherulites/radial clusters using scanningelectron microscopy (SEM), see Example 1 for additional details.

FIG. 2 shows a micrograph of spherulites/radial clusters using polarizedlight microscopy (PLM), see Example 1 for additional details.

FIG. 3A shows a micrograph of the crystal morphology as described inExample 2 (Lot J2) using scanning electron microscopy (SEM).

FIG. 3B shows a micrograph of the crystal morphology as described inExample 2 (Lot J2).

FIG. 4A shows a micrograph of the crystal morphology as described inExample 2 (Lot J1) using scanning electron microscopy (SEM).

FIG. 4B shows a micrograph of the crystal morphology as described inExample 2 (Lot J1).

FIG. 5A shows a micrograph of the crystal plate morphology as describedin Example 3 using polarized light microscopy (PLM).

FIG. 5B shows the same micrograph as in FIG. 5A of the crystal platemorphology as described in Example 3 using polarized light microscopy(PLM) but also includes an added outline of a substantiallyintact/complete free-plate having an elongated hexagonal shape.

FIG. 6 shows a differential scanning calorimetry (DSC) thermogram forLot A1 (plates) as described in Example 3.

FIG. 7 shows a differential scanning calorimetry (DSC) thermogram forLot A2 (plates) as described in Example 3.

FIG. 8 shows a differential scanning calorimetry (DSC) thermogram forLot A3 (plates) as described in Example 3.

FIG. 9 shows a differential scanning calorimetry (DSC) thermogram forLot A4 (plates) as described in Example 3.

FIG. 10 shows a differential scanning calorimetry (DSC) thermogram forLot A5 (plates) as described in Example 3.

FIG. 11 shows a micrograph of the crystal plate morphology as describedin Example 4.1, Method 1 using polarized light microscopy (PLM).

FIG. 12A shows a micrograph of the crystal plate morphology as describedin Example 4.1, Method 1 using polarized light microscopy (PLM).

FIG. 12B shows a differential scanning calorimetry (DSC) thermogram fora sample from Example 4.1, Method 1 (plates).

FIG. 13 shows a PXRD pattern overlay for Lot A5 and the sample fromExample 4.2, Method 2 showing that the sample has the same crystal phaseas the Lot A5 (plates).

FIG. 14 shows a micrograph for a sample from Example 4.2, Method 2 usingpolarized light microscopy (PLM). It was observed that the sampleconsisted predominately as plates.

FIG. 15 shows a flowchart for the preparation of core tablets of theL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid (Compound 1).

FIG. 16 shows an adsorption phase and a desorption phase from a dynamicmoisture-sorption (DMS) analysis for a representative lot having platemorphology (i.e., Lot A2).

FIG. 17 shows an adsorption/desorption cycle for a representative lothaving plate morphology (i.e., Lot A2).

FIG. 18 shows a PXRD pattern overlay for five lots (i.e., A1 to A5,plates) of the L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid (Compound 1). See Example 3 for additional details.

FIG. 19 shows a PXRD Pattern overlay for the L-arginine salt of Compound1 showing the peak intensity differences between Lot A2 (plates) and LotH2 (spherulites) indicating a higher degree of crystallinity for theplates compared to the spherulites. Also shown is the lowersample-related background scatter (i.e., a lower amorphous halocontribution) for the Lot A2 (plates).

FIG. 20 shows a micrograph for a crystalline sample from Example 7,Method 1 Step B (WO2011/094008) using polarized light microscopy (PLM).The sample was observed to have uniform radial cluster (spherulite)shaped particles with a few fine particles.

FIG. 21 shows a purification flow chart, see Example 8 for additionaldetails.

FIG. 22 shows a differential scanning calorimetry (DSC) thermogram forLot A6 (plates) as described in Example 3.

FIG. 23 shows a differential scanning calorimetry (DSC) thermogram forLot A7 (plates) as described in Example 3.

FIG. 24 shows a differential scanning calorimetry (DSC) thermogram forLot A8 (plates) as described in Example 3.

FIG. 25 shows a differential scanning calorimetry (DSC) thermogram forLot A9 (plates) as described in Example 3.

DETAILED DESCRIPTION OF THE INVENTION Definitions

For clarity and consistency, the following definitions will be usedthroughout this patent document.

The phrase “L-arginine salt of Compound 1” refers to the L-arginine saltof(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid:

The phrase “as depicted in” with reference to a Figure refers to thecrystal form and/or morphology as being characterized by graphical data“as depicted in” the Figure. Such data include, for example, powderX-ray diffractograms, differential scanning calorimetry traces, anddynamic moisture sorption graphs. The skilled person will understandthat such graphical representations of data may be subject to smallvariations, e.g., in peak relative intensities and peak positions due tofactors such as variations in instrument response and variations insample concentration and purity, which are well known to the skilledperson. Nonetheless, the skilled person would readily be capable ofcomparing the graphical data in the Figures herein with graphical datagenerated for an unknown crystal form and/or morphology (habit) andconfirm whether the two sets of graphical data are characterizing thesame crystal form (or morphology) or two different crystal forms (ormorphologies). A crystalline free-plate habit of the L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)-benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid (Compound 1) referred to herein as being characterized by graphicaldata “as depicted in” a Figure will thus be understood to include anymorphologies characterized with the graphical data having such smallvariations, as are well known to the skilled person, in comparison withthe Figure.

The term “composition” refers to a compound or salt thereof, such as,L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)-benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, in combination with at least one additional component.

The term “formulating” as used herein refers to the step(s) to transformthe bulk active pharmaceutical agent (i.e., API) into the drug substanceor drug product for use in an individual for the treatment of a disease,wherein “treatment” and “individual” has the same definitions asdescribed herein.

The phrase “free-plate habit” refers to the general shape of anindependent substantially flat crystal with a length and width of thecrystal being similar and substantially greater than the thickness andwherein the plate is not part of a radial cluster such as a spherulite.It is appreciated that due to the thin characteristic of the crystals,the phrase “free-plate habit” encompasses complete plates, substantiallycomplete plates, fragments/broken pieces of plates, and mixtures thereofthat are free or substantially free of radial clusters or spherulites.

The term “spherulite” and “radial cluster” refers to a crystal habitconsisting of thin plates or flakes that are clustered in a radialmanner around a nucleation site that exhibit a cross when viewed usingcrossed-polarized light, see FIG. 1 and FIG. 2 for micrographs ofrepresentative spherulites/radial clusters. Typically the length of thethin plates making up the radial cluster is less than 10 μm.

The term “length” in the context of a crystalline habit refers to thelongest dimension from edge to edge of a particle oriented parallel tothe ocular scale.

The term “width” in the context of a crystalline habit refers to thelongest dimension of the particle measured at right angles to thelength.

The term “individual” as used herein refers to any animal, includingmammals, preferably mice, rats, other rodents, rabbits, dogs, cats,swine, cattle, sheep, horses, or primates and most preferably humans.

The term “inert atmosphere” as used herein refers to an atmospheresubstantially free of oxygen. Examples of an inert atmosphere include,for example, argon and nitrogen.

The term “pharmaceutical composition” as used herein refers to acomposition comprising at least one active ingredient; such as Compound1 and the L-arginine salt thereof, whereby the composition is amenableto investigation for a specified pharmacological outcome in a mammal(for example, without limitation, a human) or the treatment of a diseaseor disorder as described herein. Those of ordinary skill in the art willunderstand and appreciate the techniques appropriate for determiningwhether an active ingredient has a desired efficacious outcome basedupon the needs of the artisan.

The term “treatment” or “treating” as used herein includes one or moreof the following:

(1) prevention of a disease, for example, prevention of a disease,condition or disorder in an individual that may be predisposed to thedisease, condition or disorder but does not yet experience or displaythe pathology or symptomatology of the disease;

(2) inhibition of a disease, for example, inhibition of a disease,condition or disorder in an individual that is experiencing ordisplaying the pathology or symptomatology of the disease, condition ordisorder (i.e., arresting further development of the pathology and/orsymptomatology); and

(3) amelioration of a disease, for example, amelioration of a disease,condition or disorder in an individual that is experiencing ordisplaying the pathology or symptomatology of the disease, condition ordisorder (i.e., reversing the pathology and/or symptomatology).

Whether an individual is in need of treatment is a judgment made by acaregiver (e.g. nurse practitioner, physician, physician assistant,nurse, etc. in the case of humans; veterinarian in the case of animals,including non-human mammals) that an individual or animal requires orwill benefit from treatment. This judgment is made based on a variety offactors that are in the realm of a caregiver's expertise, but thatincludes the knowledge that the individual or animal is ill, or willbecome ill, as the result of a disease, condition or disorder that istreatable by Compound 1 or a pharmaceutically acceptable salt, solvate,and hydrate thereof, such as, the L-arginine salt of Compound 1.Accordingly, Compound 1 and/or pharmaceutically acceptable salts,solvates and hydrates thereof, such as, the L-arginine salt of Compound1, can be used in a protective or preventive manner; or Compound 1 andpharmaceutically acceptable salts, solvates and hydrates thereof, suchas, the L-arginine salt of Compound 1, can be used to alleviate,inhibit, or ameliorate a disease, condition, or disorder.

The phrase “water-miscible anti-solvent” as used herein refers to awater soluble solvent in which the product, such as the L-arginine saltof(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)-benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, has limited solubility.

Chemical Group, Moiety or Radical

The term “C₂-C₄ alkanol” refers to a straight or branched carbon radicalcontaining 2 to 4 carbons bonded to an —OH group. Some embodiments are 2to 3 carbons and some embodiments are 3 to 4 carbons. Examples of aC₂-C₄ alkanol include, but are not limited to, ethanol, 1-propanol,2-propanol, 1-butanol, 2-butanol, and the like.

The term “C₁-C₆ alkyl” refers to a straight or branched carbon radicalcontaining 1 to 6 carbons. Some embodiments are 1 to 5 carbons, someembodiments are 1 to 4 carbons, some embodiments are 1 to 3 carbons andsome embodiments are 1 or 2 carbons. Examples of an alkyl include, butare not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, tert-pentyl,neo-pentyl, 1-methylbutyl [i.e., CH(CH₃)CH₂CH₂CH₃], 2-methylbutyl [i.e.,CH₂CH(CH₃)CH₂CH₃], n-hexyl and the like.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination. In addition, subcombinations of uses andmedical indications listed in the embodiments describing such uses andmedical indications described herein, are also specifically embraced bythe present invention just as if each and every subcombination of usesand medical indications was individually and explicitly recited herein.

Certain Methods of the Invention

Described herein are methods for preparing the crystalline platemorphology of the L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid using an enzymatic hydrolysis of the corresponding (R/S)-ethylester to the (R)-acid followed by a modified L-arginine salt-formingprocedure compared to what was previously disclosed in WO2011/094008 andisolating the L-arginine salt of Compound 1 as the crystallinefree-plate habit or morphology.

Accordingly, one aspect of the present invention relates to methods forpreparing a crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, the method comprising the steps of:

a) hydrolyzing a compound of Formula (IIa):

wherein R³ is C₁-C₆ alkyl; in the presence of a hydrolyzing mixturecomprising a lipase and a hydrolyzing-step solvent to form(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid;

b) adding L-arginine to a salt-forming mixture comprising(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, a water-miscible anti-solvent, and H₂O to form a first mixturecomprising L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid;

c) heating the first mixture to a first heating temperature;

d) adding a first additional amount of the water-miscible anti-solventto the first mixture to form a suspension; and

e) cooling the suspension to form the crystalline free-plate habit ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.

In some embodiments, the method for preparing a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid is conducted under an inert atmosphere. In some embodiments, themethod is conducted under an inert atmosphere comprising nitrogen orargon. In some embodiments, the method is conducted under an inertatmosphere comprising nitrogen.

Step a)—Hydrolyzing a Compound of Formula (IIa).

In some embodiments, the hydrolyzing in Step a) is conducted under aninert atmosphere. In some embodiments, the hydrolyzing in Step a) isconducted under an inert atmosphere comprising nitrogen or argon. Insome embodiments, the hydrolyzing in Step a) is conducted under an inertatmosphere comprising nitrogen.

In some embodiments, R³ is methyl or ethyl. In some embodiments, R³ isethyl.

In some embodiments, the lipase is Candida antarctica lipase B. In someembodiments, the lipase is immobilized Candida antarctica lipase B.

In some embodiments, the hydrolyzing-step solvent comprisesdimethylformamide (DMF), dimethylacetamide (DMA), dimethylsulfoxide(DMSO), tetrahydrofuran (THF), or acetonitrile. In some embodiments, thehydrolyzing-step solvent comprises acetonitrile.

In some embodiments, the compound of Formula (IIa) is:

the lipase is immobilized Candida antarctica lipase B; and thehydrolyzing-step solvent comprises acetonitrile.

In some embodiments, the hydrolyzing in Step a) is conducted in thepresence of a phosphate buffer. In some embodiments, the hydrolyzing inStep a) is conducted in the presence of a phosphate buffer at a pH ofabout 6.0 to about 9.0. In some embodiments, the hydrolyzing in Step a)is conducted in the presence of a phosphate buffer at a pH of about 7.0to about 8.5. In some embodiments, the hydrolyzing in Step a) isconducted in the presence of a phosphate buffer at a pH of about 7.3 toabout 8.3. In some embodiments, the hydrolyzing in Step a) is conductedin the presence of a phosphate buffer at a pH of about 7.6 to about 8.0.In some embodiments, the hydrolyzing in Step a) is conducted in thepresence of a phosphate buffer at a pH of about 7.8. In someembodiments, the phosphate buffer is a sodium phosphate buffer. In someembodiments, the phosphate buffer is a potassium phosphate buffer.

In some embodiments, the hydrolyzing in Step a) is conducted at atemperature of about 0° C. to about 75° C. In some embodiments, thehydrolyzing in Step a) is conducted at a temperature of about 20° C. toabout 65° C. In some embodiments, the hydrolyzing in Step a) isconducted at a temperature of about 30° C. to about 55° C. In someembodiments, the hydrolyzing in Step a) is conducted at a temperature ofabout 35° C. to about 45° C.

In some embodiments, the hydrolyzing in Step a) further comprises thestep of isolating the(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.

In some embodiments, after isolating, the(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)-benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has an enantiomeric excess of about 95% or greater. In someembodiments, after isolating, the(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)-benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has an enantiomeric excess of about 98% or greater. In someembodiments, after isolating, the(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)-benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has an enantiomeric excess of about 99% or greater.

Step b)—Adding L-Arginine to a Salt-Forming Mixture.

In some embodiments, the adding in Step b) is conducted under an inertatmosphere. In some embodiments, the adding in Step b) is conductedunder an inert atmosphere comprising argon or nitrogen. In someembodiments, the adding in Step b) is conducted under an inertatmosphere comprising nitrogen.

In some embodiments, the water-miscible anti-solvent in Step b)comprises a solvent selected from the group consisting of: acetonitrile,acetone, tetrahydrofuran, and C₂-C₄ alkanol. In some embodiments, thewater-miscible anti-solvent in Step b) comprises a solvent selected fromthe group consisting of: acetonitrile, acetone, tetrahydrofuran,ethanol, 1-propanol, 2-propanol, and 1-butanol. In some embodiments, thewater-miscible anti-solvent in Step b) comprises 2-propanol.

In some embodiments, the molar ratio between(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and L-arginine is about 1.0:0.95 to about 1.0:1.2. In someembodiments, the molar ratio between(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and L-arginine is about 1.0:1.0 to about 1.0:1.2. In someembodiments, the molar ratio between(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and L-arginine is about 1.0:1.0.

In some embodiments, the salt-forming mixture prior to the addingL-arginine is at a temperature of about 15° C. to about 83° C. In someembodiments, the salt-forming mixture prior to the adding L-arginine isat a temperature of about 18° C. to about 80° C. In some embodiments,the salt-forming mixture prior to the adding L-arginine is at atemperature of about 20° C. to about 40° C.

One aspect of the present invention allows for the L-arginine to beadded to the salt-forming mixture as a solid.

In some embodiments, the adding L-arginine in Step b) is conducted byadding L-arginine as a solid to the salt-forming mixture.

In some embodiments, the adding L-arginine in Step b) is conducted byadding L-arginine as a solid to the salt-forming mixture substantiallyall at once. The phrase “substantially all at once” or “all at once”refers to the addition of all of the L-arginine to the salt-formingmixture at one time with the only limitation that is placed on theaddition is by any limitation associated with the equipment used.

In some embodiments, the adding L-arginine in Step b) is conducted byadding L-arginine as a solid to the salt-forming mixture over a periodof 30 minutes. In some embodiments, the adding L-arginine in Step b) isconducted by adding L-arginine as a solid to the salt-forming mixtureover a period of 1 hour. In some embodiments, the adding L-arginine inStep b) is conducted by adding L-arginine as a solid to the salt-formingmixture over a period of 2 hours.

In some embodiments, the salt-forming mixture comprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.00:4.98:0.94 toabout 1.00:7.46:1.40. In some embodiments, the salt-forming mixturecomprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.00:5.29:0.99 toabout 1.00:7.15:1.35. In some embodiments, the salt-forming mixturecomprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.00:5.60:1.05 toabout 1.00:6.84:1.29. In some embodiments, the salt-forming mixturecomprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.00:5.72:1.08 toabout 1.00:6.72:1.26. In some embodiments, the salt-forming mixturecomprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.00:5.85:1.10 toabout 1.00:6.59:1.24. In some embodiments, the salt-forming mixturecomprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.00:5.97:1.12 toabout 1.00:6.47:1.22. In some embodiments, the salt-forming mixturecomprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.00:6.10:1.15 toabout 1.00:6.34:1.19. In some embodiments, the salt-forming mixturecomprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.00:6.22:1.17.

In some embodiments, the salt-forming mixture during the addingL-arginine is at a temperature of about 15° C. to about 83° C. In someembodiments, the salt-forming mixture during the adding L-arginine is ata temperature of about 18° C. to about 80° C. In some embodiments, thesalt-forming mixture during the adding L-arginine is at a temperature ofabout 20° C. to about 40° C.

One aspect of the present invention allows for the L-arginine to beadded to the salt-forming mixture as an aqueous solution.

In some embodiments, the adding L-arginine in Step b) is conducted byadding L-arginine as an aqueous solution to the salt-forming mixture.

In some embodiments, the adding L-arginine in Step b) is conducted byadding L-arginine to the salt-forming mixture wherein L-arginine is asolution of about 2.1M to about 2.3M aqueous solution at a temperatureof about 50° C. to about 75° C. In some embodiments, the addingL-arginine in Step b) is conducted by adding L-arginine to thesalt-forming mixture wherein L-arginine is a solution of about 2.26 M toabout 2.28 M aqueous solution at a temperature of about 55° C. to about65° C.

In some embodiments, after adding L-arginine as an aqueous solution inStep b) the weight ratio of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and H₂O is about 1.00:4.98:0.94 to about1.00:7.46:1.40. In some embodiments, after adding L-arginine as anaqueous solution in Step b) the weight ratio of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and H₂O is about 1.00:5.29:0.99 to about1.00:7.15:1.35. In some embodiments, after adding L-arginine as anaqueous solution in Step b) the weight ratio of(R)-2-(7-(4-cyclopentyl-3-(trifluoro-methyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and H₂O is about 1.00:5.60:1.05 to about1.00:6.84:1.29. In some embodiments, after adding L-arginine as anaqueous solution in Step b) the weight ratio of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and H₂O is about 1.00:5.72:1.08 to about1.00:6.72:1.26. In some embodiments, after adding L-arginine as anaqueous solution in Step b) the weight ratio of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclo-penta[b]indol-3-yl)aceticacid, 2-propanol, and H₂O is about 1.00:5.85:1.10 to about1.00:6.59:1.24. In some embodiments, after adding L-arginine as anaqueous solution in Step b) the weight ratio of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and H₂O is about 1.00:5.97:1.12 to about1.00:6.47:1.22. In some embodiments, after adding L-arginine as anaqueous solution in Step b) the weight ratio of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and H₂O is about 1.00:6.10:1.15 to about1.00:6.34:1.19. In some embodiments, after adding L-arginine as anaqueous solution in Step b) the weight ratio of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyl-oxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and H₂O is about 1.00:6.22:1.17.

In some embodiments, Step b) is conducted at a stir rate of about 100rpm to about 200 rpm. In some embodiments, Step b) is conducted at astir rate of about 125 rpm to about 175 rpm. In some embodiments, Stepb) is conducted at a stir rate of about 150 rpm.

Step c)—Heating the First Mixture to a First Heating Temperature.

In some embodiments, the first heating temperature is about 20° C. toabout 83° C. In some embodiments, the first heating temperature is about20° C. to about 55° C. In some embodiments, the first heatingtemperature is about 25° C. to about 45° C. In some embodiments, thefirst heating temperature is about 50° C. to about 83° C. In someembodiments, the first heating temperature is about 60° C. to about 80°C. In some embodiments, the first heating temperature is about 70° C. toabout 80° C.

In some embodiments, the first mixture is substantially a homogeneoussolution.

In some embodiments, Step c) is conducted at a stir rate of about 100rpm to about 200 rpm. In some embodiments, Step c) is conducted at astir rate of about 125 rpm to about 175 rpm. In some embodiments, Stepc) is conducted at a stir rate of about 150 rpm.

Step d)—Adding a First Additional Amount of the Water-MiscibleAnti-Solvent to the First Mixture to Form a Suspension.

In some embodiments, the water-miscible anti-solvent in Step b)comprises a solvent selected from the group consisting of: acetonitrile,acetone, tetrahydrofuran, and C₂-C₄ alkanol. In some embodiments, thewater-miscible anti-solvent in Step b) comprises a solvent selected fromthe group consisting of: acetonitrile, acetone, tetrahydrofuran,ethanol, 1-propanol, 2-propanol, and 1-butanol. In some embodiments, thewater-miscible anti-solvent in Step b) comprises 2-propanol.

In some embodiments, the weight ratio of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:5.46 toabout 1.00:8.20. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:5.81 toabout 1.00:7.86. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:6.15 toabout 1.00:7.51. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:6.28 toabout 1.00:7.38. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:6.42 toabout 1.00:7.24. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:6.56 toabout 1.00:7.10. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:6.69 toabout 1.00:6.97. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:6.83.

In some embodiments, adding the first additional amount of 2-propanol tothe first mixture is conducted at a rate to complete the addition inabout 5.00 hours to about 10.00 hours. In some embodiments, adding thefirst additional amount of 2-propanol to the first mixture is conductedat a rate to complete the addition in about 6.75 hours to about 8.25hours. In some embodiments, adding the first additional amount of2-propanol to the first mixture is conducted at a rate to complete theaddition in about 6.90 hours to about 8.10 hours. In some embodiments,adding the first additional amount of 2-propanol to the first mixture isconducted at a rate to complete the addition in about 7.05 hour to about7.95 hour. In some embodiments, adding the first additional amount of2-propanol to the first mixture is conducted at a rate to complete theaddition in about 7.20 hours to about 7.80 hours. In some embodiments,adding the first additional amount of 2-propanol to the first mixture isconducted at a rate to complete the addition in about 7.35 hours toabout 7.65 hours. In some embodiments, adding the first additionalamount of 2-propanol to the first mixture is conducted at a rate tocomplete the addition in about 7.50 hours.

In some embodiments, the first heating temperature is maintained duringadding the first additional amount of 2-propanol to the first mixture.

In some embodiments, after adding the first additional amount of2-propanol to the first mixture in Step c), the method further comprisesforming a second mixture while maintaining the first heatingtemperature, wherein the second mixture is formed prior to forming thesuspension.

In some embodiments, the method further comprises cooling the secondmixture to a first cooling temperature followed by heating to a secondheating temperature to form the suspension.

In some embodiments, cooling the second mixture to the first coolingtemperature is conducted at a rate of about 8.80° C./hour to about14.40° C./hour. In some embodiments, cooling the second mixture to thefirst cooling temperature is conducted at a rate of about 9.35° C./hourto about 13.80° C./hour. In some embodiments, cooling the second mixtureto the first cooling temperature is conducted at a rate of about 9.90°C./hour to about 13.20° C./hour. In some embodiments, cooling the secondmixture to the first cooling temperature is conducted at a rate of about10.45° C./hour to about 12.60° C./hour. In some embodiments, cooling thesecond mixture to the first cooling temperature is conducted at a rateof about 10° C./hour to about 12° C./hour.

In some embodiments, the first cooling temperature is about 15° C. toabout 40° C. In some embodiments, the first cooling temperature is about20° C. to about 30° C. In some embodiments, the first coolingtemperature is about 22° C. to about 24° C.

In some embodiments, the first cooling temperature is maintained for atleast 1 hour prior to heating to the second heating temperature.

In some embodiments, the second heating temperature is about 65° C. toabout 83° C. In some embodiments, the second heating temperature isabout 70° C. to about 80° C. In some embodiments, the second heatingtemperature is about 70° C. to about 75° C.

In some embodiments, after heating to the second heating temperature,the second heating temperature is maintained for at least 30 minutes.

In some embodiments, Step d) is conducted at a stir rate of about 100rpm to about 200 rpm. In some embodiments, Step d) is conducted at astir rate of about 125 rpm to about 175 rpm. In some embodiments, Stepd) is conducted at a stir rate of about 150 rpm.

Step e)—Cooling the Suspension to Form the Crystalline Free-Plate Habitof L-Arginine Salt of(R)-2-(7-(4-Cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticAcid.

In some embodiments, cooling the suspension in Step e) is conducted at arate of about 8.8° C./hour to about 14.4° C./hour. In some embodiments,cooling the suspension in Step e) is conducted at a rate of about 9.4°C./hour to about 13.8° C./hour. In some embodiments, cooling thesuspension in Step e) is conducted at a rate of about 9.9° C./hour toabout 13.2° C./hour. In some embodiments, cooling the suspension in Stepe) is conducted at a rate of about 10.5° C./hour to about 12.6° C./hour.In some embodiments, cooling the suspension in Step e) is conducted at arate of about 10° C./hour to about 12° C./hour.

In some embodiments, after cooling in Step e) the temperature of thesuspension is about 15° C. to about 40° C. In some embodiments, aftercooling in Step e) the temperature of the suspension is about 20° C. toabout 30° C. In some embodiments, after cooling in Step e) thetemperature of the suspension is about 22° C. to about 24° C.

In some embodiments, Step e) is conducted at a stir rate of about 100rpm to about 200 rpm. In some embodiments, Step e) is conducted at astir rate of about 125 rpm to about 175 rpm. In some embodiments, Stepe) is conducted at a stir rate of about 150 rpm.

In some embodiments, the method further comprises the step of isolatingthe crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.

In some embodiments, the step of isolating comprises filtering thecrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid from the suspension. In some embodiments, the step of isolatingcomprises filtering the crystalline free-plate habit of L-arginine saltof(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid from the suspension and drying the crystalline free-plate habit ofL-arginine salt at a reduced pressure.

In some embodiments, after filtering, the crystalline free-plate habitof L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has an onset temperature of 205.0° C. to 208.5° C. as determined bydifferential scanning calorimetry at a scan rate of 10° C./minute. Insome embodiments, after filtering, the crystalline free-plate habit ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has an onset temperature of 205.5° C. to 208.5° C. as determined bydifferential scanning calorimetry at a scan rate of 10° C./minute. Insome embodiments, after filtering, the crystalline free-plate habit ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has an onset temperature of 206.5° C. to 208.5° C. as determined bydifferential scanning calorimetry at a scan rate of 10° C./minute. Insome embodiments, after filtering, the crystalline free-plate habit ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has an onset temperature of 207.0° C. to 208.1° C. as determined bydifferential scanning calorimetry at a scan rate of 10° C./minute. Insome embodiments, after filtering, the crystalline free-plate habit ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a differential scanning calorimetry trace conducted at a scanrate of 10° C./minute comprising an endotherm substantially as depictedin any one of FIGS. 6 to 10. In some embodiments, after filtering, thecrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a differential scanning calorimetry trace conducted at a scanrate of 10° C./minute comprising an endotherm substantially as depictedin any one of FIGS. 6 to 10 and FIGS. 22 to 25. In some embodiments,after filtering, the crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a dynamic moisture sorption (DMS) profile with an adsorptionphase from 30% RH to 90% RH wherein the crystalline free-plate habitgains about 0.3% weight or less at 90% RH. In some embodiments, afterfiltering, the crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a dynamic moisture sorption (DMS) profile with an adsorptionphase from 30% RH to 90% RH wherein the crystalline free-plate habitgains about 0.2% weight or less at 90% RH. The DMS features reportedherein can also vary by plus or minus about 0.15% weight change (i.e.,±0.15% weight change).

In some embodiments, after filtering, the crystalline free-plate habitof L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a total impurity profile by achiral HPLC of less than or equalto 0.5% by area. In some embodiments, after filtering, the crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a total impurity profile by achiral HPLC of less than or equalto 0.3% by area. In some embodiments, after filtering, the crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a total impurity profile by achiral HPLC of less than or equalto 0.2% by area.

In some embodiments, the method further comprises the step offormulating the crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid to form a pharmaceutical composition.

In some embodiments, the method further comprises the step of admixingthe crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid with a pharmaceutical excipient to form a pharmaceuticalcomposition.

One aspect of the present invention relates to methods for preparing acrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, the method comprising the steps of:

a) hydrolyzing a compound of Formula (IIa):

wherein R³ is C₁-C₆ alkyl; in the presence of a hydrolyzing mixturecomprising a lipase and a hydrolyzing-step solvent to form(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid;

b) adding L-arginine to a salt-forming mixture comprising(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and H₂O to form a first mixture comprising L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid;

c) heating the first mixture to a first heating temperature;

d) adding a first additional amount of the 2-propanol to the firstmixture to form a suspension; and

e) cooling the suspension to form the crystalline free-plate habit ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid. Further description of the embodiments for these methods asrelated to Steps a) to e) can be found herein, for example, see Steps a)to e) respectively, supra.

Also described herein are methods for preparing a crystalline free-platehabit of the L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid starting from(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.

Accordingly, one aspect of the present invention relates to methods forpreparing a crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, the method comprising the steps of:

a) adding L-arginine to a salt-forming mixture comprising(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, a water-miscible anti-solvent, and H₂O to form a first mixturecomprising L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid;

b) heating the first mixture to a first temperature;

c) adding a first additional amount of the water-miscible anti-solventto the first mixture to form a suspension; and

d) cooling the suspension to form the crystalline free-plate habit ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid. Further description of the embodiments for these methods asrelated to Steps a) to d) can be found herein, for example, see Steps b)to e) respectively, supra.

Another aspect of the present invention relates to methods for preparinga crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, the method comprising the steps of:

a) adding L-arginine to a salt-forming mixture comprising(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and H₂O to form a first mixture comprising L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid;

b) heating the first mixture to a first temperature;

c) adding a first additional amount of 2-propanol to the first mixtureto form a suspension; and

d) cooling the suspension to form the crystalline free-plate habit ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid. Further description of the embodiments for these methods asrelated to Steps a) to d) can be found herein, for example, see Steps b)to e) respectively, supra.

One aspect of the present invention relates to methods for preparing acrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, the method comprising the steps of:

a) hydrolyzing a compound of Formula (IIa):

wherein R³ is C₁-C₆ alkyl; in the presence of a hydrolyzing mixturecomprising a lipase and a hydrolyzing-step solvent to form(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid;

b) forming a first mixture comprising L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, a water-miscible anti-solvent, and H₂O;

c) heating the first mixture to a first heating temperature to form asecond mixture;

d) adding a first additional amount of the water-miscible anti-solventto the second mixture while maintaining the first heating temperature toform a suspension;

e) cooling the suspension to a first cooling temperature and thereafterheating to a second heating temperature;

f) cycling Step e) optionally one or more times, wherein the firstcooling temperature at each cycle may be the same or different and thesecond heating temperature at each cycle may be the same or different;and

g) cooling the suspension to a final cooling temperature to form thecrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.

In some embodiments, the method for preparing a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid is conducted under an inert atmosphere. In some embodiments, themethod is conducted under an inert atmosphere comprising nitrogen orargon. In some embodiments, the method is conducted under an inertatmosphere comprising nitrogen.

Step a)—Hydrolyzing a Compound of Formula (IIa).

In some embodiments, the hydrolyzing in Step a) is conducted under aninert atmosphere. In some embodiments, the hydrolyzing in Step a) isconducted under an inert atmosphere comprising nitrogen or argon. Insome embodiments, the hydrolyzing in Step a) is conducted under an inertatmosphere comprising nitrogen.

In some embodiments, R³ is methyl or ethyl. In some embodiments, R³ isethyl.

In some embodiments, the lipase is Candida Antarctica lipase B. In someembodiments, the lipase is immobilized Candida Antarctica lipase B.

In some embodiments, the hydrolyzing-step solvent comprisesdimethylformamide (DMF), dimethylacetamide (DMA), dimethylsulfoxide(DMSO), tetrahydrofuran (THF), or acetonitrile. In some embodiments, thehydrolyzing-step solvent comprises acetonitrile.

In some embodiments, the compound of Formula (IIa) is:

the lipase is immobilized Candida Antarctica lipase B; and thehydrolyzing-step solvent comprises acetonitrile.

In some embodiments, the hydrolyzing is conducted in the presence of aphosphate buffer. In some embodiments, the hydrolyzing is conducted inthe presence of a phosphate buffer at a pH of about 6.0 to about 9.0. Insome embodiments, the hydrolyzing is conducted in the presence of aphosphate buffer at a pH of about 6.9 to about 8.1. In some embodiments,the hydrolyzing is conducted in the presence of a phosphate buffer at apH of about 7.0 to about 8.5. In some embodiments, the hydrolyzing isconducted in the presence of a phosphate buffer at a pH of about 7.3 toabout 8.3. In some embodiments, the hydrolyzing is conducted in thepresence of a phosphate buffer at a pH of about 7.6 to about 8.0. Insome embodiments, the hydrolyzing is conducted in the presence of aphosphate buffer at a pH of about 7.8. In some embodiments, thephosphate buffer is a sodium phosphate buffer. In some embodiments, thephosphate buffer is a potassium phosphate buffer.

In some embodiments, the hydrolyzing is conducted at a temperature ofabout 0° C. to about 75° C. In some embodiments, the hydrolyzing isconducted at a temperature of about 20° C. to about 65° C. In someembodiments, the hydrolyzing is conducted at a temperature of about 30°C. to about 55° C. In some embodiments, the hydrolyzing is conducted ata temperature of about 35° C. to about 45° C.

In some embodiments, the hydrolyzing further comprises the step ofisolating the(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.

In some embodiments, after the isolating, the(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has an enantiomeric excess of about 95% or greater. In someembodiments, after the isolating, the(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has an enantiomeric excess of about 98% or greater. In someembodiments, after the isolating, the(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has an enantiomeric excess of about 99% or greater.

In some embodiments, after hydrolyzing in Step a), the(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid is not isolated.

In some embodiments, after hydrolyzing in Step a), the(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid is present in an amount of at least 40% as determined by HPLC. Insome embodiments, after the hydrolyzing, the(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid is present in an amount of at least 45% as determined by HPLC.

Step b)—Forming a First Mixture.

In some embodiments, forming a first mixture in Step b) is conductedunder an inert atmosphere. In some embodiments, forming a first mixturein Step b) is conducted under an inert atmosphere comprising argon ornitrogen. In some embodiments, forming a first mixture in Step b) isconducted under an inert atmosphere comprising nitrogen.

It is understood that forming a first mixture comprising L-arginine saltof(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, a water-miscible anti-solvent, and H₂O, may be performed in anymanner routine to the skilled artisan.

By way of example, forming the first mixture may comprise forming theL-arginine salt by reacting L-arginine with(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid; and mixing the L-arginine salt, the water-miscible anti-solvent,and H₂O.

By way of example, forming the first mixture may comprise forming amixture of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, the water-miscible anti-solvent and H₂O; and reacting theL-arginine with(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid to form the first mixture.

By way of example, forming the first mixture may comprise forming amixture of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and the water-miscible anti-solvent; and reacting the L-arginine asan aqueous slurry or solution with(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid to form the first mixture.

By way of example, forming the first mixture may comprise forming amixture of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, the water-miscible anti-solvent and the L-arginine; and adding H₂Oto form the first mixture.

Accordingly, in some embodiments, forming the first mixture in Step b)comprises the step of adding L-arginine and H₂O, either together orseparately in any order, to a salt-forming mixture comprising(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and the water-miscible anti-solvent to form the first mixture.

In some embodiments, the adding L-arginine and H₂O is conducted under aninert atmosphere. In some embodiments, the adding L-arginine and H₂O isconducted under an inert atmosphere comprising argon or nitrogen. Insome embodiments, the adding L-arginine and H₂O is conducted under aninert atmosphere comprising nitrogen.

In some embodiments, the water-miscible anti-solvent comprises a solventselected from the group consisting of: acetonitrile, acetone,tetrahydrofuran, and C₂-C₄ alkanol. In some embodiments, thewater-miscible anti-solvent comprises a solvent selected from the groupconsisting of: acetonitrile, acetone, tetrahydrofuran, ethanol,1-propanol, 2-propanol, and 1-butanol. In some embodiments, thewater-miscible anti-solvent comprises 2-propanol.

In some embodiments, prior to adding the L-arginine and H₂O, thesalt-forming mixture comprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid and 2-propanol in a weight ratio of about 1.0:3.0 to about1.0:11.0. In some embodiments, prior to adding the L-arginine and H₂O,the salt-forming mixture comprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid and 2-propanol in a weight ratio of about 1.0:4.0 to about1.0:10.0. In some embodiments, prior to adding the L-arginine and H₂O,the salt-forming mixture comprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid and 2-propanol in a weight ratio of about 1.0:5.0 to about 1.0:9.0.In some embodiments, prior to adding the L-arginine and H₂O, thesalt-forming mixture comprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid and 2-propanol in a weight ratio of about 1.0:6.0 to about 1.0:8.0.

In some embodiments, the molar ratio between(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and L-arginine is about 1.0:0.8 to about 1.0:1.2.

In some embodiments, the molar ratio between(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and L-arginine is about 1.0:0.9 to about 1.0:1.2. In someembodiments, the molar ratio between(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and L-arginine is about 1.0:1.0 to about 1.0:1.2. In someembodiments, the molar ratio between(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and L-arginine is about 1.0:0.93 to about 1.0:1.01. In someembodiments, the molar ratio between(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and L-arginine is about 1.0:0.93 to about 1.0:0.97. In someembodiments, the molar ratio between(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and L-arginine is about 1.0:1.0. In some embodiments, the molarratio between(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and L-arginine is about 1.0:0.95.

In some embodiments, the weight ratio of L-arginine and H₂O is about1.0:0.4 to about 1.0:2.3. In some embodiments, the weight ratio ofL-arginine and H₂O is about 1.0:1.6 to about 1.0:2.1. In someembodiments, the weight ratio of L-arginine and H₂O is about 1.0:0.8 toabout 1.0:1.9. In some embodiments, the weight ratio of L-arginine andH₂O is about 1.0:1.0 to about 1.0:1.7. In some embodiments, the weightratio of L-arginine and H₂O is about 1.0:1.1 to about 1.0:1.6. In someembodiments, the weight ratio of L-arginine and H₂O is about 1.0:1.2 toabout 1.0:1.5.

In some embodiments, the salt-forming mixture prior to the addingL-arginine is at a temperature of about 15° C. to about 83° C. In someembodiments, the salt-forming mixture prior to the adding L-arginine isat a temperature of about 18° C. to about 80° C. In some embodiments,the salt-forming mixture prior to the adding L-arginine is at atemperature of about 20° C. to about 40° C. In some embodiments, thesalt-forming mixture prior to the adding L-arginine is at a temperatureof about 18° C. to about 30° C.

In some embodiments, the adding L-arginine is conducted by addingL-arginine as an aqueous slurry to the salt-forming mixture.

In some embodiments, the adding L-arginine is conducted by addingL-arginine as a solid to the salt-forming mixture.

In some embodiments, the adding L-arginine to the salt-forming mixtureis conducted by adding L-arginine as a solid to the salt-forming mixturesubstantially all at once. The phrase “substantially all at once” or“all at once” refers to the addition of all of the L-arginine to thesalt-forming mixture at one time with the only limitation that is placedon the addition is by any limitation associated with the equipment used.

In some embodiments, the adding L-arginine to the salt-forming mixtureis conducted by adding L-arginine as a solid to the salt-forming mixtureover a period of about 30 minutes. In some embodiments, the addingL-arginine to the salt-forming mixture is conducted by adding L-arginineas a solid to the salt-forming mixture over a period of about 1 hour. Insome embodiments, the adding L-arginine to the salt-forming mixture isconducted by adding L-arginine as a solid to the salt-forming mixtureover a period of about 2 hours.

In some embodiments, the salt-forming mixture comprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.0:3.0:0.05 toabout 1.0:11.0:1.0. In some embodiments, the salt-forming mixturecomprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.0:4.0:0.1 toabout 1.0:10.0:0.9. In some embodiments, the salt-forming mixturecomprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.0:5.0:0.15 toabout 1.0:9.0:0.8. In some embodiments, the salt-forming mixturecomprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.0:6.0:0.25 toabout 1.0:8.0:0.7.

In some embodiments, the salt-forming mixture comprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.00:4.98:0.94 toabout 1.00:7.46:1.40, about 1.00:5.29:0.99 to about 1.00:7.15:1.35,about 1.00:5.60:1.05 to about 1.00:6.84:1.29, about 1.00:5.72:1.08 toabout 1.00:6.72:1.26, about 1.00:5.85:1.10 to about 1.00:6.59:1.24,about 1.00:5.97:1.12 to about 1.00:6.47:1.22, about 1.00:6.10:1.15 toabout 1.00:6.34:1.19, or about 1.00:6.22:1.17.

In some embodiments, the salt-forming mixture during the addingL-arginine is at a temperature of about 15° C. to about 83° C. In someembodiments, the salt-forming mixture during the adding L-arginine is ata temperature of about 18° C. to about 80° C. In some embodiments, thesalt-forming mixture during the adding L-arginine is at a temperature ofabout 20° C. to about 40° C. In some embodiments, the salt-formingmixture during the adding L-arginine is at a temperature of about 18° C.to about 30° C.

In some embodiments, the adding L-arginine is conducted by addingL-arginine as an aqueous solution to the salt-forming mixture.

In some embodiments, the adding L-arginine is conducted by addingL-arginine to the salt-forming mixture wherein L-arginine is a solutionof about 2.1M to about 2.3M aqueous solution at a temperature of about50° C. to about 75° C.

In some embodiments, forming the first mixture in Step b) is conductedat a stir rate of about 100 rpm to about 200 rpm. In some embodiments,forming the first mixture in Step b) is conducted at a stir rate ofabout 125 rpm to about 175 rpm. In some embodiments, forming the firstmixture in Step b) is conducted at a stir rate of about 150 rpm.

Step c)—Heating the First Mixture to a First Heating Temperature to Forma Second Mixture.

In some embodiments, the first heating temperature is about 20° C. toabout 85° C. In some embodiments, the first heating temperature is about20° C. to about 83° C. In some embodiments, the first heatingtemperature is about 20° C. to about 55° C. In some embodiments, thefirst heating temperature is about 25° C. to about 45° C. In someembodiments, the first heating temperature is about 50° C. to about 83°C. In some embodiments, the first heating temperature is about 60° C. toabout 80° C. In some embodiments, the first heating temperature is about70° C. to about 80° C. In some embodiments, the first heatingtemperature is about 79° C. to about 85° C.

In some embodiments, an optional amount of H₂O is added to the secondmixture.

In some embodiments, when the optional amount of H₂O is added to thesecond mixture, the weight amount of water added is about 0.4 to about2.3 times the weight of L-arginine originally added in forming the firstmixture.

In some embodiments, when the optional amount of H₂O is added to thesecond mixture, the weight amount of water added is about 1.0:1.6 toabout 1.0:2.1 times the weight of L-arginine originally added in formingthe first mixture. In some embodiments, when water is added to thesecond mixture, the weight amount of water added is about 1.0:0.8 toabout 1.0:1.9 times the weight of L-arginine originally added in formingthe first mixture. In some embodiments, when water is added to thesecond mixture, the weight amount of water added is about 1.0:1.0 toabout 1.0:1.7 times the weight of L-arginine originally added in formingthe first mixture. In some embodiments, when water is added to thesecond mixture, the weight amount of water added is about 1.0:1.1 toabout 1.0:1.6 times the weight of L-arginine originally added in formingthe first mixture. In some embodiments, when water is added to thesecond mixture, the weight amount of water added is about 1.0:1.2 toabout 1.0:1.5 times the weight of L-arginine originally added in formingthe first mixture.

In some embodiments, the second mixture is substantially a homogeneoussolution.

In some embodiments, Step c) is conducted at a stir rate of about 100rpm to about 200 rpm. In some embodiments, Step c) is conducted at astir rate of about 125 rpm to about 175 rpm. In some embodiments, Stepc) is conducted at a stir rate of about 150 rpm.

Step d)—Adding a First Additional Amount of the Water-MiscibleAnti-Solvent to the Second Mixture while Maintaining the First HeatingTemperature to Form a Suspension.

In some embodiments, the weight ratio of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:5.46 toabout 1.00:8.20.

In some embodiments, the weight ratio of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:4.70 toabout 1.00:7.50. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:5.20 toabout 1.00:7.00. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:5.70 toabout 1.00:6.50. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:5.90 toabout 1.00:6.30. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:5.95 toabout 1.00:6.25. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:6.00 toabout 1.00:6.20.

In some embodiments, the weight ratio of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:5.46 toabout 1.00:8.20, about 1.00:5.81 to about 1.00:7.86, about 1.00:6.15 toabout 1.00:7.51, about 1.00:6.28 to about 1.00:7.38, about 1.00:6.42 toabout 1.00:7.24, about 1.00:6.56 to about 1.00:7.10, about 1.00:6.69 toabout 1.00:6.97, or about 1.00:6.83.

In some embodiments, the first additional amount of the water-miscibleanti-solvent is added during a first time point and a second time point.

In some embodiments, about 5% to about 15% of the first additionalamount of the water-miscible anti-solvent is added at the first timepoint.

In some embodiments, about 7% to about 13% of the first additionalamount of the water-miscible anti-solvent is added at the first timepoint.

In some embodiments, about 8% to about 12% of the first additionalamount of the water-miscible anti-solvent is added at the first timepoint. In some embodiments, about 9% to about 11% of the firstadditional amount of the water-miscible anti-solvent is added at thefirst time point.

In some embodiments, the first additional amount of the water-miscibleanti-solvent is added at the first time point to form a cloudy mixture.

In some embodiments, prior to the second time point, a seed crystal ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid is optionally added.

In some embodiments, the first additional amount of the water-miscibleanti-solvent is added at the second time point at a rate to complete theaddition in about 1.00 hour or greater. In some embodiments, the firstadditional amount of the water-miscible anti-solvent is added at thesecond time point at a rate to complete the addition in about 1.00 hourto about 6.00 hours. In some embodiments, the first additional amount ofthe water-miscible anti-solvent is added at the second time point at arate to complete the addition in about 1.00 hour to about 5.00 hours. Insome embodiments, the first additional amount of the water-miscibleanti-solvent is added at the second time point at a rate to complete theaddition in about 1.00 hour to about 4.00 hours. In some embodiments,the first additional amount of the water-miscible anti-solvent is addedat the second time point at a rate to complete the addition in about1.00 hour to about 3.00 hours. In some embodiments, the first additionalamount of the water-miscible anti-solvent is added at the second timepoint at a rate to complete the addition in about 1.00 hour to about2.00 hours.

In some embodiments, the first heating temperature is maintained duringthe addition of the first additional amount of 2-propanol to the secondmixture.

In some embodiments, Step d) is conducted at a stir rate of about 100rpm to about 200 rpm. In some embodiments, Step d) is conducted at astir rate of about 125 rpm to about 175 rpm. In some embodiments, Stepd) is conducted at a stir rate of about 150 rpm.

Step e)—Cooling the Suspension to a First Cooling Temperature andThereafter Heating to a Second Heating Temperature.

In some embodiments, cooling the suspension to the first coolingtemperature in Step e) is conducted at a rate of about 8.80° C./hour toabout 14.40° C./hour. In some embodiments, cooling the suspension to thefirst cooling temperature in Step e) is conducted at a rate of about9.35° C./hour to about 13.80° C./hour. In some embodiments, cooling thesuspension to the first cooling temperature in Step e) is conducted at arate of about 9.90° C./hour to about 13.20° C./hour. In someembodiments, cooling the suspension to the first cooling temperature inStep e) is conducted at a rate of about 10.45° C./hour to about 12.60°C./hour. In some embodiments, cooling the suspension to the firstcooling temperature in Step e) is conducted at a rate of about 10°C./hour to about 12° C./hour. In some embodiments, cooling thesuspension to the first cooling temperature in Step e) is conducted at arate of about 9° C./hour to about 11° C./hour. In some embodiments,cooling the suspension to the first cooling temperature in Step e) isconducted at a rate of about 9.5° C./hour to about 10.5° C./hour.

In some embodiments, the first cooling temperature in Step e) is about15° C. to about 40° C. In some embodiments, the first coolingtemperature in Step e) is about 20° C. to about 30° C. In someembodiments, the first cooling temperature in Step e) is about 22° C. toabout 24° C. In some embodiments, the first cooling temperature in Stepe) is about 18° C. to about 22° C.

In some embodiments, the first cooling temperature is maintained for atleast 1 hour prior to heating to the second heating temperature.

In some embodiments, the second heating temperature in Step e) is about65° C. to about 83° C.

In some embodiments, the second heating temperature in Step e) is about70° C. to about 80° C. In some embodiments, the second heatingtemperature in Step e) is about 70° C. to about 75° C. In someembodiments, the second heating temperature in Step e) is about 69° C.to about 73° C.

In some embodiments, after heating to the second heating temperature,the second heating temperature is maintained for at least 30 minutes.

In some embodiments, Step e) is conducted at a stir rate of about 100rpm to about 200 rpm. In some embodiments, Step e) is conducted at astir rate of about 125 rpm to about 175 rpm. In some embodiments, Stepe) is conducted at a stir rate of about 150 rpm.

Step f)—Cycling Step e) Optionally One or More Times.

It is understood that when cycling Step e) more than once that the firstcooling temperature at each cycle may be the same or different and saidsecond heating temperature at each cycle may be the same or different.

Two Cycles

In some embodiments, the cycling in Step f) comprises the cycling Stepe) two times.

In some embodiments, cycling Step e) two times comprises cooling thesuspension to a first cycling cooling temperature, heating thesuspension to a first cycling heating temperature, cooling thesuspension to a second cycling cooling temperature, and heating thesuspension to a second cycling heating temperature.

In some embodiments, the first cycling cooling temperature is about 16°C. to about 26° C., the first heating cycling temperature is about 55°C. to about 65° C., the second cycling cooling temperature is about 26°C. to about 36° C., and the second cycling heating temperature is about45° C. to about 55° C. In some embodiments, the first cycling coolingtemperature is about 19° C. to about 23° C., the first heating cyclingtemperature is about 58° C. to about 62° C., the second cycling coolingtemperature is about 29° C. to about 33° C., and the second cyclingheating temperature is about 48° C. to about 52° C.

In some embodiments, cooling to each cooling temperature is conducted ata substantially different cooling rate. In some embodiments, cooling toeach cooling temperature is conducted at a different cooling rate.

In some embodiments, the cycling in Step f) comprises the cycling Stepe) two times. In some embodiments, cooling to the first cycling coolingtemperature is conducted at a rate of about 10.0° C./hour to about 15.0°C./hour. In some embodiments, cooling to the first cycling coolingtemperature is conducted at a rate of about 11.0° C./hour to about 14.0°C./hour. In some embodiments, cooling to the first cycling coolingtemperature is conducted at a rate of about 12.0° C./hour to about 13.0°C./hour. In some embodiments, cooling to the first cycling coolingtemperature is conducted at a rate of about 12.5° C./hour.

In some embodiments, the cycling in Step f) comprises the cycling Stepe) two times. In some embodiments, cooling to the second cycling coolingtemperature is conducted at a rate of about 7.5° C./hour to about 12.5°C./hour. In some embodiments, cooling to the second cycling coolingtemperature is conducted at a rate of about 8.5° C./hour to about 11.5°C./hour. In some embodiments, cooling to the second cycling coolingtemperature is conducted at a rate of about 9.5° C./hour to about 10.5°C./hour. In some embodiments, cooling to the second cycling coolingtemperature is conducted at a rate of about 10.0° C./hour.

Three Cycles

In some embodiments, the cycling in Step f) comprises the cycling Stepe) three times.

In some embodiments, cycling Step e) three times comprises: cooling thesuspension to a first cycling cooling temperature, heating thesuspension to a first cycling heating temperature, cooling thesuspension to a second cycling cooling temperature, heating thesuspension to a second cycling heating temperature, cooling thesuspension to a third cycling cooling temperature, and heating thesuspension to a third cycling heating temperature.

In some embodiments, the first cycling cooling temperature is about 16°C. to about 26° C., the first heating cycling temperature is about 66°C. to about 76° C., the second cycling cooling temperature is about 16°C. to about 26° C., the second cycling heating temperature is about 55°C. to about 65° C., the third cycling cooling temperature is about 26°C. to about 36° C., and the third cycling heating temperature is about45° C. to about 55° C. In some embodiments, the first cycling coolingtemperature is about 19° C. to about 23° C., the first heating cyclingtemperature is about 69° C. to about 73° C., the second cycling coolingtemperature is about 19° C. to about 23° C., the second cycling heatingtemperature is about 58° C. to about 62° C., the third cycling coolingtemperature is about 29° C. to about 33° C., and the third cyclingheating temperature is about 48° C. to about 52° C.

In some embodiments, cooling to each cooling temperature is conducted ata substantially different cooling rate. In some embodiments, cooling toeach cooling temperature is conducted at a different cooling rate.

In some embodiments, cooling to the first cycling cooling temperatureand the second cycling cooling temperature are each conducted atsubstantially the same cooling rate.

In some embodiments, the cycling in Step f) comprises the cycling Stepe) three times. In some embodiments, cooling to the first cyclingcooling temperature is conducted at a rate of about 10.0° C./hour toabout 15.0° C./hour. In some embodiments, cooling to the first cyclingcooling temperature is conducted at a rate of about 11.0° C./hour toabout 14.0° C./hour. In some embodiments, cooling to the first cyclingcooling temperature is conducted at a rate of about 12.0° C./hour toabout 13.0° C./hour. In some embodiments, cooling to the first cyclingcooling temperature is conducted at a rate of about 12.5° C./hour.

In some embodiments, the cycling in Step f) comprises the cycling Stepe) three times. In some embodiments, cooling to the second cyclingcooling temperature is conducted at a rate of about 10.0° C./hour toabout 15.0° C./hour. In some embodiments, cooling to the second cyclingcooling temperature is conducted at a rate of about 11.0° C./hour toabout 14.0° C./hour. In some embodiments, cooling to the second cyclingcooling temperature is conducted at a rate of about 12.0° C./hour toabout 13.0° C./hour. In some embodiments, cooling to the first cyclingcooling temperature is conducted at a rate of about 12.5° C./hour.

In some embodiments, the cycling in Step f) comprises the cycling Stepe) three times. In some embodiments, cooling to the third cyclingcooling temperature is conducted at a rate of about 7.5° C./hour toabout 12.5° C./hour. In some embodiments, cooling to the third cyclingcooling temperature is conducted at a rate of about 8.5° C./hour toabout 11.5° C./hour. In some embodiments, cooling to the third cyclingcooling temperature is conducted at a rate of about 9.5° C./hour toabout 10.5° C./hour. In some embodiments, cooling to the second cyclingcooling temperature is conducted at a rate of about 10.0° C./hour.

Four Cycles

In some embodiments, the cycling in Step f) comprises the cycling Stepe) four times.

In some embodiments, cycling Step e) four times comprises: cooling thesuspension to a first cycling cooling temperature, heating thesuspension to a first cycling heating temperature, cooling thesuspension to a second cycling cooling temperature, heating thesuspension to a second cycling heating temperature, cooling thesuspension to a third cycling cooling temperature, heating thesuspension to a third cycling heating temperature, cooling thesuspension to a fourth cycling cooling temperature, and heating thesuspension to a fourth cycling heating temperature.

In some embodiments, the first cycling cooling temperature is about 16°C. to about 26° C., the first heating cycling temperature is about 66°C. to about 76° C., the second cycling cooling temperature is about 16°C. to about 26° C., the second cycling heating temperature is about 66°C. to about 76° C., the third cycling cooling temperature is about 16°C. to about 26° C., the third cycling heating temperature is about 55°C. to about 65° C., the fourth cycling cooling temperature is about 26°C. to about 36° C., and the fourth cycling heating temperature is about45° C. to about 55° C. In some embodiments, the first cycling coolingtemperature is about 19° C. to about 23° C., the first heating cyclingtemperature is about 69° C. to about 73° C., the second cycling coolingtemperature is about 19° C. to about 23° C., the second cycling heatingtemperature is about 69° C. to about 73° C., the third cycling coolingtemperature is about 19° C. to about 23° C., the third cycling heatingtemperature is about 58° C. to about 62° C., the fourth cycling coolingtemperature is about 29° C. to about 33° C., and the fourth cyclingheating temperature is about 48° C. to about 52° C.

In some embodiments, cooling to each cooling temperature is conducted ata substantially different cooling rate. In some embodiments, cooling toeach cooling temperature is conducted at a different cooling rate.

In some embodiments, cooling to the first cycling cooling temperature,the second cycling cooling temperature, and the third cycling coolingtemperature are each conducted at substantially the same cooling rate.

In some embodiments, the cycling in Step f) comprises the cycling Stepe) four times. In some embodiments, cooling to the first cycling coolingtemperature is conducted at a rate of about 10.0° C./hour to about 15.0°C./hour. In some embodiments, cooling to the first cycling coolingtemperature is conducted at a rate of about 11.0° C./hour to about 14.0°C./hour. In some embodiments, cooling to the first cycling coolingtemperature is conducted at a rate of about 12.0° C./hour to about 13.0°C./hour. In some embodiments, cooling to the first cycling coolingtemperature is conducted at a rate of about 12.5° C./hour.

In some embodiments, the cycling in Step f) comprises the cycling Stepe) four times. In some embodiments, cooling to the second cyclingcooling temperature is conducted at a rate of about 10.0° C./hour toabout 15.0° C./hour. In some embodiments, cooling to the second cyclingcooling temperature is conducted at a rate of about 11.0° C./hour toabout 14.0° C./hour. In some embodiments, cooling to the second cyclingcooling temperature is conducted at a rate of about 12.0° C./hour toabout 13.0° C./hour. In some embodiments, cooling to the first cyclingcooling temperature is conducted at a rate of about 12.5° C./hour.

In some embodiments, the cycling in Step f) comprises the cycling Stepe) four times. In some embodiments, cooling to the third cycling coolingtemperature is conducted at a rate of about 10.0° C./hour to about 15.0°C./hour. In some embodiments, cooling to the third cycling coolingtemperature is conducted at a rate of about 11.0° C./hour to about 14.0°C./hour. In some embodiments, cooling to the third cycling coolingtemperature is conducted at a rate of about 12.0° C./hour to about 13.0°C./hour. In some embodiments, cooling to the first cycling coolingtemperature is conducted at a rate of about 12.5° C./hour.

In some embodiments, the cycling in Step f) comprises the cycling Stepe) four times. In some embodiments, cooling to the fourth cyclingcooling temperature is conducted at a rate of about 7.5° C./hour toabout 12.5° C./hour. In some embodiments, cooling to the fourth cyclingcooling temperature is conducted at a rate of about 8.5° C./hour toabout 11.5° C./hour. In some embodiments, cooling to the fourth cyclingcooling temperature is conducted at a rate of about 9.5° C./hour toabout 10.5° C./hour. In some embodiments, cooling to the second cyclingcooling temperature is conducted at a rate of about 10.0° C./hour.

In some embodiments, Step f) is conducted at a stir rate of about 100rpm to about 200 rpm. In some embodiments, Step f) is conducted at astir rate of about 125 rpm to about 175 rpm. In some embodiments, Stepf) is conducted at a stir rate of about 150 rpm.

Step g)—Cooling the Suspension to a Final Cooling Temperature to Formthe Crystalline Free-Plate Habit of L-Arginine Salt of(R)-2-(7-(4-Cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticAcid.

In some embodiments, cooling the suspension in Step g) is conducted at arate of about 7.5° C./hour to about 12.5° C./hour. In some embodiments,cooling the suspension in Step g) is conducted at a rate of about 8.5°C./hour to about 11.5° C./hour. In some embodiments, cooling thesuspension in Step g) is conducted at a rate of about 9.5° C./hour toabout 10.5° C./hour. In some embodiments, cooling the suspension in Stepg) is conducted at a rate of about 10.0° C./hour.

In some embodiments, cooling the suspension in Step g) is conducted at arate of about 8.8° C./hour to about 14.4° C./hour. In some embodiments,cooling the suspension in Step g) is conducted at a rate of about 9.4°C./hour to about 13.8° C./hour. In some embodiments, cooling thesuspension in Step g) is conducted at a rate of about 9.9° C./hour toabout 13.2° C./hour. In some embodiments, cooling the suspension in Stepg) is conducted at a rate of about 10.5° C./hour to about 12.6° C./hour.In some embodiments, cooling the suspension in Step g) is conducted at arate of about 10° C./hour to about 12° C./hour.

In some embodiments, after cooling in Step g) the temperature of thesuspension is about 15° C. to about 40° C. In some embodiments, aftercooling in Step g) the temperature of the suspension is about 20° C. toabout 30° C. In some embodiments, after cooling in Step g) thetemperature of the suspension is about 22° C. to about 24° C. In someembodiments, after cooling in Step g) the temperature of said suspensionis about 18° C. to about 22° C.

In some embodiments, Step g) is conducted at a stir rate of about 100rpm to about 200 rpm. In some embodiments, Step g) is conducted at astir rate of about 125 rpm to about 175 rpm. In some embodiments, Stepg) is conducted at a stir rate of about 150 rpm.

In some embodiments, the method further comprises the step of isolatingthe crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.

In some embodiments, the step of isolating comprises filtering thecrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid from the suspension. In some embodiments, the step of isolatingcomprises filtering the crystalline free-plate habit of L-arginine saltof(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid from the suspension and drying the crystalline free-plate habit ofL-arginine salt at a reduced pressure.

In some embodiments, after filtering, the crystalline free-plate habitof L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has any one or more of the characteristics as described herein. Forexample, In some embodiments, after filtering, the crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has an onset temperature of 205.0° C. to 208.5° C. as determined bydifferential scanning calorimetry at a scan rate of 10° C./minute. Insome embodiments, after filtering, the crystalline free-plate habit ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has an onset temperature of 205.5° C. to 208.5° C. as determined bydifferential scanning calorimetry at a scan rate of 10° C./minute. Insome embodiments, after filtering, the crystalline free-plate habit ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has an onset temperature of 206.5° C. to 208.5° C. as determined bydifferential scanning calorimetry at a scan rate of 10° C./minute. Insome embodiments, after filtering, the crystalline free-plate habit ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has an onset temperature of 207.0° C. to 208.1° C. as determined bydifferential scanning calorimetry at a scan rate of 10° C./minute. Insome embodiments, after filtering, the crystalline free-plate habit ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a differential scanning calorimetry trace conducted at a scanrate of 10° C./minute comprising an endotherm substantially as depictedin any one of FIGS. 6 to 10. In some embodiments, after filtering, thecrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a differential scanning calorimetry trace conducted at a scanrate of 10° C./minute comprising an endotherm substantially as depictedin any one of FIGS. 6 to 10 and FIGS. 22 to 25. In some embodiments,after filtering, the crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a dynamic moisture sorption (DMS) profile with an adsorptionphase from 30% RH to 90% RH wherein the crystalline free-plate habitgains about 0.3% weight or less at 90% RH. In some embodiments, afterfiltering, the crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a dynamic moisture sorption (DMS) profile with an adsorptionphase from 30% RH to 90% RH wherein the crystalline free-plate habitgains about 0.2% weight or less at 90% RH. The DMS features reportedherein can also vary by plus or minus about 0.15% weight change (i.e.,±0.15% weight change).

In some embodiments, after filtering, the crystalline free-plate habitof L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a total impurity profile by achiral HPLC of less than or equalto 0.5% by area. In some embodiments, after filtering, the crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a total impurity profile by achiral HPLC of less than or equalto 0.3% by area. In some embodiments, after filtering, the crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a total impurity profile by achiral HPLC of less than or equalto 0.2% by area.

In some embodiments, the method further comprises the step offormulating the crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid to form a pharmaceutical composition.

In some embodiments, the method further comprises the step of admixingthe crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid with a pharmaceutical excipient to form a pharmaceuticalcomposition.

In some embodiments, the method further comprises the step of admixingthe crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid with a pharmaceutical excipient to form a pharmaceuticalcomposition suitable for oral, rectal, nasal, topical, buccal,sub-lingual, or vaginal, or in a form suitable for administration byinhalation, insufflation, or by a transdermal patch. In someembodiments, the pharmaceutical composition is suitable for oraladministration.

Also described herein are methods for preparing a crystalline free-platehabit of the L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid starting from a different morphology, such as, but not limited to,spherulites/radial clusters, fine particles, agglomerates, flakes, or amixture thereof.

Accordingly, one aspect of the present invention relates to methods forpreparing a crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, the method comprising the steps of:

a) heating a first mixture comprising L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, a water-miscible anti-solvent, and water to a first heatingtemperature to form a second mixture;

b) cooling the second mixture to a first cooling temperature followed byadding a first additional amount of the water-miscible anti-solvent tothe second mixture while maintaining the first cooling temperature andthereafter heating to a second heating temperature to form a suspension;

c) cycling Step b) optionally once or twice; and

d) cooling the suspension to a second cooling temperature to form thecrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.

In some embodiments, the water-miscible anti-solvent comprises a solventselected from the group consisting of: acetonitrile, acetone,tetrahydrofuran, and C₂-C₄ alkanol. In some embodiments, thewater-miscible anti-solvent comprises a solvent selected from the groupconsisting of: acetonitrile, acetone, tetrahydrofuran, ethanol,1-propanol, 2-propanol, and 1-butanol. In some embodiments, thewater-miscible anti-solvent comprises 2-propanol.

In some embodiments, the L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)-benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid in Step a) is a spherulite, a radial cluster, a fine particle, anagglomerate, a flake, or a mixture thereof.

In some embodiments, the L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)-benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid in Step a) is a crystalline spherulite habit or a mixturecomprising a crystalline spherulite habit.

In some embodiments, the method for preparing a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid is conducted under an inert atmosphere. In some embodiments, themethod is conducted under an inert atmosphere comprising nitrogen orargon. In some embodiments, the method is conducted under an inertatmosphere comprising nitrogen.

Step a)—Heating a First Mixture.

In some embodiments, the first mixture comprises L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.00:3.35:0.75 toabout 1.00:5.03:1.13. In some embodiments, the first mixture comprisesL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.00:3.56:0.80 toabout 1.00:4.82:1.08. In some embodiments, the first mixture comprisesL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.00:3.77:0.85 toabout 1.00:4.61:1.03. In some embodiments, the first mixture comprisesL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.00:3.85:0.86 toabout 1.00:4.52:1.02. In some embodiments, the first mixture comprisesL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.00:3.94:0.88 toabout 1.00:4.44:1.00. In some embodiments, the first mixture comprisesL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.00:4.02:0.90 toabout 1.00:4.36:0.98. In some embodiments, the first mixture comprisesL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.00:4.11:0.92 toabout 1.00:4.27:0.96. In some embodiments, the first mixture comprisesL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.00:4.19:0.94.

In some embodiments, the first mixture is at a temperature of about 10°C. to about 30° C. prior to heating to the first heating temperature. Insome embodiments, the first mixture is at a temperature of about 15° C.to about 25° C. prior to heating to the first heating temperature.

In some embodiments, the first heating temperature is about 60° C. toabout 83° C. In some embodiments, the first heating temperature is about70° C. to about 83° C. In some embodiments, the first heatingtemperature is about 75° C. to about 80° C.

In some embodiments, after heating to the first heating temperature thesecond mixture is substantially a homogeneous solution.

In some embodiments, Step a) is conducted at a stir rate of about 50 rpmto about 250 rpm. In some embodiments, Step a) is conducted at a stirrate of about 75 rpm to about 150 rpm.

Step b)—Cooling the Second Mixture to a First Cooling Temperature.

In some embodiments, the first cooling temperature is about 50° C. toabout 70° C. In some embodiments, the first cooling temperature is about60° C. to about 70° C. In some embodiments, the first coolingtemperature is about 63° C. to about 67° C.

In some embodiments, the first cooling temperature is maintained for atleast 30 minutes.

In some embodiments, adding the first additional amount of 2-propanol tothe second mixture is conducted at a rate of about 107.20 g/minute toabout 160.80 g/minute. In some embodiments, adding the first additionalamount of 2-propanol to the second mixture is conducted at a rate ofabout 113.90 g/minute to about 154.10 g/minute. In some embodiments,adding the first additional amount of 2-propanol to the second mixtureis conducted at a rate of about 120.60 g/minute to about 147.40g/minute. In some embodiments, adding the first additional amount of2-propanol to the second mixture is conducted at a rate of about 127.30g/minute to about 140.70 g/minute. In some embodiments, adding the firstadditional amount of 2-propanol to the second mixture is conducted at arate of about 134 g/minute.

In some embodiments, the weight ratio of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:0.98 toabout 1.00:1.47. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:1.04 toabout 1.00:1.41. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:1.10 toabout 1.00:1.35. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:1.13 toabout 1.00:1.32. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:1.15 toabout 1.00:1.30. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:1.18 toabout 1.00:1.27. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:1.20 toabout 1.00:1.25. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:1.23.

In some embodiments, after adding the first additional amount of2-propanol, the first cooling temperature is maintained for at least 1hour prior to heating to the second heating temperature.

In some embodiments, the second heating temperature is about 65° C. toabout 83° C. In some embodiments, the second heating temperature isabout 70° C. to about 80° C. In some embodiments, the second heatingtemperature is about 70° C. to about 75° C.

In some embodiments, the second heating temperature is at a temperatureto retain the suspension.

In some embodiments, after heating to the second heating temperature,the second heating temperature is maintained for at least 30 minuteswhile retaining the suspension.

In some embodiments, Step b) is conducted at a stir rate of about 50 rpmto about 150 rpm. In some embodiments, Step b) is conducted at a stirrate of about 70 rpm to about 110 rpm.

Step c)—Cycling Step b).

In some embodiments, cycling Step b) is conducted once.

In some embodiments, cycling Step b) is conducted once, wherein thecycling conditions are substantially the same conditions as used forStep b).

In some embodiments, cycling Step b) is conducted once, wherein thecycling conditions are different from the conditions used for Step b);it is understood that the selected cycling conditions are still withinthe embodiments as described herein for Step b).

In some embodiments, cycling Step b) is conducted twice.

In some embodiments, cycling Step b) is conducted twice, wherein thecycling conditions for the first cycle and the second cycle aresubstantially the same conditions as used for Step b).

In some embodiments, cycling Step b) is conducted twice, wherein atleast one of the conditions for the first cycle, the second cycle, orStep b) is different; it is understood that whether or not the firstcycle and/or the second cycle is different from each other or one orboth of the cycles are different from Step b) that each of selectedconditions for the first cycle and the second cycle are still within theembodiments as described herein for Step b).

Step d)—Cooling the Suspension to a Second Cooling Temperature to Formthe Crystalline Free-Plate Habit of L-Arginine Salt of(R)-2-(7-(4-Cyclopentyl-3-(trifluoro-methyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticAcid.

In some embodiments, the second cooling temperature is about 50° C. toabout 70° C. In some embodiments, the second cooling temperature isabout 60° C. to about 70° C. In some embodiments, the second coolingtemperature is about 63° C. to about 67° C.

In some embodiments, the second cooling temperature is maintained for atleast 30 minutes.

In some embodiments, Step d) is conducted at a stir rate of about 25 rpmto about 105 rpm. In some embodiments, Step d) is conducted at a stirrate of about 45 rpm to about 85 rpm.

In some embodiments, after cooling to the second cooling temperature,the method further comprises the step of cooling the suspension to athird cooling temperature.

In some embodiments, after cooling to the second cooling temperature,the method further comprises the step of cooling the suspension to athird cooling temperature of about 10° C. to about 30° C. In someembodiments, after cooling to the second cooling temperature, the methodfurther comprises the step of cooling the suspension to a third coolingtemperature of about 15° C. to about 25° C.

In some embodiments, the cooling rate from the second coolingtemperature to the third cooling temperature is about 5° C. per hour toabout 15° C. per hour. In some embodiments, the cooling rate from thesecond cooling temperature to the third cooling temperature is about 8°C. per hour to about 12° C. per hour. In some embodiments, the coolingrate from the second cooling temperature to the third coolingtemperature is about 10° C. per hour.

In some embodiments, the method further comprises the step of isolatingthe crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.

In some embodiments, the step of isolating comprises filtering thecrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid from the suspension. In some embodiments, the step of isolatingcomprises filtering the crystalline free-plate habit of L-arginine saltof(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid from the suspension and drying the crystalline free-plate habit ofL-arginine salt at a reduced pressure.

In some embodiments, after filtering, the crystalline free-plate habitof L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has an onset temperature of 205.0° C. to 208.5° C. as determined bydifferential scanning calorimetry at a scan rate of 10° C./minute. Insome embodiments, after filtering, the crystalline free-plate habit ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has an onset temperature of 205.5° C. to 208.5° C. as determined bydifferential scanning calorimetry at a scan rate of 10° C./minute. Insome embodiments, after filtering, the crystalline free-plate habit ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has an onset temperature of 206.5° C. to 208.5° C. as determined bydifferential scanning calorimetry at a scan rate of 10° C./minute. Insome embodiments, after filtering, the crystalline free-plate habit ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has an onset temperature of 207.0° C. to 208.1° C. as determined bydifferential scanning calorimetry at a scan rate of 10° C./minute. Insome embodiments, after filtering, the crystalline free-plate habit ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a differential scanning calorimetry trace conducted at a scanrate of 10° C./minute comprising an endotherm substantially as depictedin any one of FIGS. 6 to 10. In some embodiments, after filtering, thecrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a differential scanning calorimetry trace conducted at a scanrate of 10° C./minute comprising an endotherm substantially as depictedin any one of FIGS. 6 to 10 and FIGS. 22 to 25. In some embodiments,after filtering, the crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a dynamic moisture sorption (DMS) profile with an adsorptionphase from 30% RH to 90% RH wherein the crystalline free-plate habitgains about 0.3% weight or less at 90% RH. In some embodiments, afterfiltering, the crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a dynamic moisture sorption (DMS) profile with an adsorptionphase from 30% RH to 90% RH wherein the crystalline free-plate habitgains about 0.2% weight or less at 90% RH. The DMS features reportedherein can also vary by plus or minus about 0.15% weight change (i.e.,±0.15% weight change).

In some embodiments, after filtering, the crystalline free-plate habitof L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a total impurity profile by achiral HPLC of less than or equalto 0.5% by area. In some embodiments, after filtering, the crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a total impurity profile by achiral HPLC of less than or equalto 0.3% by area. In some embodiments, after filtering, the crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a total impurity profile by achiral HPLC of less than or equalto 0.2% by area.

In some embodiments, the method further comprises the step offormulating the crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid to form a pharmaceutical composition.

In some embodiments, the method further comprises the step of admixingthe crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid with a pharmaceutical excipient to form a pharmaceuticalcomposition.

One aspect of the present invention relates to methods for preparing acrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, the method comprising the steps of:

a) forming a first mixture comprising L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, a water-miscible anti-solvent, and H₂O;

b) heating the first mixture to a first heating temperature to form asecond mixture;

c) adding a first additional amount of the water-miscible anti-solventto the second mixture while maintaining the first heating temperature toform a suspension;

d) cooling the suspension to a first cooling temperature and thereafterheating to a second heating temperature;

e) cycling Step d) optionally one or more times, wherein the firstcooling temperature at each cycle may be the same or different and thesecond heating temperature at each cycle may be the same or different;and

f) cooling the suspension to a final cooling temperature to form thecrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.

Step a)—Forming a First Mixture.

In some embodiments, forming a first mixture in Step a) is conductedunder an inert atmosphere. In some embodiments, forming a first mixturein Step a) is conducted under an inert atmosphere comprising argon ornitrogen. In some embodiments, forming a first mixture in Step a) isconducted under an inert atmosphere comprising nitrogen.

It is understood that forming a first mixture comprising L-arginine saltof(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, a water-miscible anti-solvent, and H₂O, may be performed in anymanner routine to the skilled artisan, such as, the examples describedsupra.

In some embodiments, forming the first mixture in Step a) comprises thestep of adding L-arginine and H₂O, either together or separately in anyorder, to a salt-forming mixture comprising(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and the water-miscible anti-solvent to form the first mixture.

In some embodiments, the adding L-arginine and H₂O is conducted under aninert atmosphere. In some embodiments, the adding L-arginine and H₂O isconducted under an inert atmosphere comprising argon or nitrogen. Insome embodiments, the adding L-arginine and H₂O is conducted under aninert atmosphere comprising nitrogen.

In some embodiments, the water-miscible anti-solvent comprises a solventselected from the group consisting of: acetonitrile, acetone,tetrahydrofuran, and C₂-C₄ alkanol. In some embodiments, thewater-miscible anti-solvent comprises a solvent selected from the groupconsisting of: acetonitrile, acetone, tetrahydrofuran, ethanol,1-propanol, 2-propanol, and 1-butanol. In some embodiments, thewater-miscible anti-solvent comprises 2-propanol.

In some embodiments, prior to adding the L-arginine and H₂O, thesalt-forming mixture comprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid and 2-propanol in a weight ratio of about 1.0:3.0 to about1.0:11.0. In some embodiments, prior to adding the L-arginine and H₂O,the salt-forming mixture comprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid and 2-propanol in a weight ratio of about 1.0:4.0 to about1.0:10.0. In some embodiments, prior to adding the L-arginine and H₂O,the salt-forming mixture comprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid and 2-propanol in a weight ratio of about 1.0:5.0 to about 1.0:9.0.In some embodiments, prior to adding the L-arginine and H₂O, thesalt-forming mixture comprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid and 2-propanol in a weight ratio of about 1.0:6.0 to about 1.0:8.0.

In some embodiments, the molar ratio between(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and L-arginine is about 1.0:0.8 to about 1.0:1.2.

In some embodiments, the molar ratio between(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and L-arginine is about 1.0:0.9 to about 1.0:1.2. In someembodiments, the molar ratio between(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and L-arginine is about 1.0:1.0 to about 1.0:1.2. In someembodiments, the molar ratio between(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and L-arginine is about 1.0:0.93 to about 1.0:1.01. In someembodiments, the molar ratio between(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and L-arginine is about 1.0:0.93 to about 1.0:0.97. In someembodiments, the molar ratio between(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and L-arginine is about 1.0:1.0. In some embodiments, the molarratio between(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and L-arginine is about 1.0:0.95.

In some embodiments, the weight ratio of L-arginine and H₂O is about1.0:0.4 to about 1.0:2.3. In some embodiments, the weight ratio ofL-arginine and H₂O is about 1.0:1.6 to about 1.0:2.1. In someembodiments, the weight ratio of L-arginine and H₂O is about 1.0:0.8 toabout 1.0:1.9. In some embodiments, the weight ratio of L-arginine andH₂O is about 1.0:1.0 to about 1.0:1.7. In some embodiments, the weightratio of L-arginine and H₂O is about 1.0:1.1 to about 1.0:1.6. In someembodiments, the weight ratio of L-arginine and H₂O is about 1.0:1.2 toabout 1.0:1.5.

In some embodiments, the salt-forming mixture prior to the addingL-arginine is at a temperature of about 15° C. to about 83° C. In someembodiments, the salt-forming mixture prior to the adding L-arginine isat a temperature of about 18° C. to about 80° C. In some embodiments,the salt-forming mixture prior to the adding L-arginine is at atemperature of about 20° C. to about 40° C. In some embodiments, thesalt-forming mixture prior to the adding L-arginine is at a temperatureof about 18° C. to about 30° C.

In some embodiments, the adding L-arginine is conducted by addingL-arginine as an aqueous slurry to the salt-forming mixture.

In some embodiments, the adding L-arginine is conducted by addingL-arginine as a solid to the salt-forming mixture.

In some embodiments, the adding L-arginine to the salt-forming mixtureis conducted by adding L-arginine as a solid to the salt-forming mixturesubstantially all at once. The phrase “substantially all at once” or“all at once” refers to the addition of all of the L-arginine to thesalt-forming mixture at one time with the only limitation that is placedon the addition is by any limitation associated with the equipment used.

In some embodiments, the adding L-arginine to the salt-forming mixtureis conducted by adding L-arginine as a solid to the salt-forming mixtureover a period of about 30 minutes. In some embodiments, the addingL-arginine to the salt-forming mixture is conducted by adding L-arginineas a solid to the salt-forming mixture over a period of about 1 hour. Insome embodiments, the adding L-arginine to the salt-forming mixture isconducted by adding L-arginine as a solid to the salt-forming mixtureover a period of about 2 hours.

In some embodiments, the salt-forming mixture comprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.0:3.0:0.05 toabout 1.0:11.0:1.0. In some embodiments, the salt-forming mixturecomprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.0:4.0:0.1 toabout 1.0:10.0:0.9. In some embodiments, the salt-forming mixturecomprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.0:5.0:0.15 toabout 1.0:9.0:0.8. In some embodiments, the salt-forming mixturecomprises(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydro-cyclopenta[b]indol-3-yl)aceticacid, 2-propanol, and water in a weight ratio of about 1.0:6.0:0.25 toabout 1.0:8.0:0.7.

In some embodiments, the salt-forming mixture during the addingL-arginine is at a temperature of about 15° C. to about 83° C. In someembodiments, the salt-forming mixture during the adding L-arginine is ata temperature of about 18° C. to about 80° C. In some embodiments, thesalt-forming mixture during the adding L-arginine is at a temperature ofabout 20° C. to about 40° C. In some embodiments, the salt-formingmixture during the adding L-arginine is at a temperature of about 18° C.to about 30° C.

In some embodiments, the salt-forming mixture during the addingL-arginine is at a temperature of about 15° C. to about 83° C. In someembodiments, the salt-forming mixture during the adding L-arginine is ata temperature of about 18° C. to about 80° C. In some embodiments, thesalt-forming mixture during the adding L-arginine is at a temperature ofabout 20° C. to about 40° C. In some embodiments, the salt-formingmixture during the adding L-arginine is at a temperature of about 18° C.to about 30° C.

In some embodiments, the adding L-arginine is conducted by addingL-arginine as an aqueous solution to the salt-forming mixture.

In some embodiments, the adding L-arginine is conducted by addingL-arginine to the salt-forming mixture wherein L-arginine is a solutionof about 2.1M to about 2.3M aqueous solution at a temperature of about50° C. to about 75° C.

In some embodiments, forming the first mixture in Step a) is conductedat a stir rate of about 100 rpm to about 200 rpm. In some embodiments,forming the first mixture in Step a) is conducted at a stir rate ofabout 125 rpm to about 175 rpm. In some embodiments, forming the firstmixture in Step a) is conducted at a stir rate of about 150 rpm.

Step b)—Heating the First Mixture to a First Heating Temperature to Forma Second Mixture.

In some embodiments, the first heating temperature is about 20° C. toabout 85° C. In some embodiments, the first heating temperature is about20° C. to about 83° C. In some embodiments, the first heatingtemperature is about 20° C. to about 55° C. In some embodiments, thefirst heating temperature is about 25° C. to about 45° C. In someembodiments, the first heating temperature is about 50° C. to about 83°C. In some embodiments, the first heating temperature is about 60° C. toabout 80° C. In some embodiments, the first heating temperature is about70° C. to about 80° C. In some embodiments, the first heatingtemperature is about 79° C. to about 85° C.

In some embodiments, an optional amount of H₂O is added to the secondmixture.

In some embodiments, when water is added to the second mixture, theweight amount of water added is about 0.4 to about 2.3 times the weightof L-arginine originally added in forming the first mixture.

In some embodiments, when water is added to the second mixture, theweight amount of water added is about 1.0:1.6 to about 1.0:2.1 times theweight of L-arginine originally added in forming the first mixture. Insome embodiments, when water is added to the second mixture, the weightamount of water added is about 1.0:0.8 to about 1.0:1.9 times the weightof L-arginine originally added in forming the first mixture. In someembodiments, when water is added to the second mixture, the weightamount of water added is about 1.0:1.0 to about 1.0:1.7 times the weightof L-arginine originally added in forming the first mixture. In someembodiments, when water is added to the second mixture, the weightamount of water added is about 1.0:1.1 to about 1.0:1.6 times the weightof L-arginine originally added in forming the first mixture. In someembodiments, when water is added to the second mixture, the weightamount of water added is about 1.0:1.2 to about 1.0:1.5 times the weightof L-arginine originally added in forming the first mixture.

In some embodiments, the second mixture is substantially a homogeneoussolution.

In some embodiments, Step b) is conducted at a stir rate of about 100rpm to about 200 rpm. In some embodiments, Step b) is conducted at astir rate of about 125 rpm to about 175 rpm. In some embodiments, Stepb) is conducted at a stir rate of about 150 rpm.

Step c)—Adding a First Additional Amount of the Water-MiscibleAnti-Solvent to the Second Mixture while Maintaining the First HeatingTemperature to Form a Suspension.

In some embodiments, the weight ratio of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:5.46 toabout 1.00:8.20.

In some embodiments, the weight ratio of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:4.70 toabout 1.00:7.50. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:5.20 toabout 1.00:7.00. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:5.70 toabout 1.00:6.50. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:5.90 toabout 1.00:6.30. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:5.95 toabout 1.00:6.25. In some embodiments, the weight ratio of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:6.00 toabout 1.00:6.20.

In some embodiments, the weight ratio of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid to the first additional amount of 2-propanol is about 1.00:5.46 toabout 1.00:8.20, about 1.00:5.81 to about 1.00:7.86, about 1.00:6.15 toabout 1.00:7.51, about 1.00:6.28 to about 1.00:7.38, about 1.00:6.42 toabout 1.00:7.24, about 1.00:6.56 to about 1.00:7.10, about 1.00:6.69 toabout 1.00:6.97, or about 1.00:6.83.

In some embodiments, the first additional amount of the water-miscibleanti-solvent is added during a first time point and a second time point.

In some embodiments, about 5% to about 15% of the first additionalamount of the water-miscible anti-solvent is added at the first timepoint. In some embodiments, about 7% to about 13% of the firstadditional amount of the water-miscible anti-solvent is added at thefirst time point. In some embodiments, about 8% to about 12% of thefirst additional amount of the water-miscible anti-solvent is added atthe first time point. In some embodiments, about 9% to about 11% of thefirst additional amount of the water-miscible anti-solvent is added atthe first time point.

In some embodiments, the first additional amount of the water-miscibleanti-solvent is added at the first time point to form a cloudy mixture.

In some embodiments, prior to the second time point, a seed crystal ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid is optionally added.

In some embodiments, the first additional amount of the water-miscibleanti-solvent is added at the second time point at a rate to complete theaddition in about 1.00 hour or greater. In some embodiments, the firstadditional amount of the water-miscible anti-solvent is added at thesecond time point at a rate to complete the addition in about 1.00 hourto about 6.00 hours. In some embodiments, the first additional amount ofthe water-miscible anti-solvent is added at the second time point at arate to complete the addition in about 1.00 hour to about 5.00 hours. Insome embodiments, the first additional amount of the water-miscibleanti-solvent is added at the second time point at a rate to complete theaddition in about 1.00 hour to about 4.00 hours. In some embodiments,the first additional amount of the water-miscible anti-solvent is addedat the second time point at a rate to complete the addition in about1.00 hour to about 3.00 hours. In some embodiments, the first additionalamount of the water-miscible anti-solvent is added at the second timepoint at a rate to complete the addition in about 1.00 hour to about2.00 hours.

In some embodiments, the first heating temperature is maintained duringthe addition of the first additional amount of 2-propanol to the secondmixture.

In some embodiments, Step c) is conducted at a stir rate of about 100rpm to about 200 rpm. In some embodiments, Step c) is conducted at astir rate of about 125 rpm to about 175 rpm. In some embodiments, Stepc) is conducted at a stir rate of about 150 rpm.

Step d)—Cooling the Suspension to a First Cooling Temperature andThereafter Heating to a Second Heating Temperature.

In some embodiments, cooling the suspension to the first coolingtemperature in Step d) is conducted at a rate of about 8.80° C./hour toabout 14.40° C./hour. In some embodiments, cooling the suspension to thefirst cooling temperature in Step d) is conducted at a rate of about9.35° C./hour to about 13.80° C./hour. In some embodiments, cooling thesuspension to the first cooling temperature in Step d) is conducted at arate of about 9.90° C./hour to about 13.20° C./hour. In someembodiments, cooling the suspension to the first cooling temperature inStep d) is conducted at a rate of about 10.45° C./hour to about 12.60°C./hour. In some embodiments, cooling the suspension to the firstcooling temperature in Step d) is conducted at a rate of about 10°C./hour to about 12° C./hour. In some embodiments, cooling thesuspension to the first cooling temperature in Step d) is conducted at arate of about 9° C./hour to about 11° C./hour. In some embodiments,cooling the suspension to the first cooling temperature in Step d) isconducted at a rate of about 9.5° C./hour to about 10.5° C./hour.

In some embodiments, the first cooling temperature in Step d) is about15° C. to about 40° C. In some embodiments, the first coolingtemperature in Step d) is about 20° C. to about 30° C. In someembodiments, the first cooling temperature in Step d) is about 22° C. toabout 24° C. In some embodiments, the first cooling temperature in Stepd) is about 18° C. to about 22° C.

In some embodiments, the first cooling temperature is maintained for atleast 1 hour prior to heating to the second heating temperature.

In some embodiments, the second heating temperature in Step d) is about65° C. to about 83° C.

In some embodiments, the second heating temperature in Step d) is about70° C. to about 80° C. In some embodiments, the second heatingtemperature in Step d) is about 70° C. to about 75° C. In someembodiments, the second heating temperature in Step d) is about 69° C.to about 73° C.

In some embodiments, after heating to the second heating temperature,the second heating temperature is maintained for at least 30 minutes.

In some embodiments, Step d) is conducted at a stir rate of about 100rpm to about 200 rpm. In some embodiments, Step d) is conducted at astir rate of about 125 rpm to about 175 rpm. In some embodiments, Stepd) is conducted at a stir rate of about 150 rpm.

Step e)—Cycling Step d) Optionally One or More Times.

It is understood that when cycling Step d) more than once that the firstcooling temperature at each cycle may be the same or different and thesecond heating temperature at each cycle may be the same or different.

Two Cycles

In some embodiments, the cycling in Step e) comprises the cycling Stepd) two times.

In some embodiments, cycling Step d) two times comprises cooling thesuspension to a first cycling cooling temperature, heating thesuspension to a first cycling heating temperature, cooling thesuspension to a second cycling cooling temperature, and heating thesuspension to a second cycling heating temperature.

In some embodiments, the first cycling cooling temperature is about 16°C. to about 26° C., the first heating cycling temperature is about 55°C. to about 65° C., the second cycling cooling temperature is about 26°C. to about 36° C., and the second cycling heating temperature is about45° C. to about 55° C. In some embodiments, the first cycling coolingtemperature is about 19° C. to about 23° C., the first heating cyclingtemperature is about 58° C. to about 62° C., the second cycling coolingtemperature is about 29° C. to about 33° C., and the second cyclingheating temperature is about 48° C. to about 52° C.

In some embodiments, cooling to each cooling temperature is conducted ata substantially different cooling rate. In some embodiments, cooling toeach cooling temperature is conducted at a different cooling rate.

In some embodiments, the cycling in Step e) comprises the cycling Stepd) two times. In some embodiments, cooling to the first cycling coolingtemperature is conducted at a rate of about 10.0° C./hour to about 15.0°C./hour. In some embodiments, cooling to the first cycling coolingtemperature is conducted at a rate of about 11.0° C./hour to about 14.0°C./hour. In some embodiments, cooling to the first cycling coolingtemperature is conducted at a rate of about 12.0° C./hour to about 13.0°C./hour. In some embodiments, cooling to the first cycling coolingtemperature is conducted at a rate of about 12.5° C./hour.

In some embodiments, the cycling in Step e) comprises the cycling Stepd) two times. In some embodiments, cooling to the second cycling coolingtemperature is conducted at a rate of about 7.5° C./hour to about 12.5°C./hour. In some embodiments, cooling to the second cycling coolingtemperature is conducted at a rate of about 8.5° C./hour to about 11.5°C./hour. In some embodiments, cooling to the second cycling coolingtemperature is conducted at a rate of about 9.5° C./hour to about 10.5°C./hour. In some embodiments, cooling to the second cycling coolingtemperature is conducted at a rate of about 10.0° C./hour.

Three Cycles

In some embodiments, the cycling in Step e) comprises the cycling Stepd) three times.

In some embodiments, cycling Step d) three times comprises: cooling thesuspension to a first cycling cooling temperature, heating thesuspension to a first cycling heating temperature, cooling thesuspension to a second cycling cooling temperature, heating thesuspension to a second cycling heating temperature, cooling thesuspension to a third cycling cooling temperature, and heating thesuspension to a third cycling heating temperature.

In some embodiments, the first cycling cooling temperature is about 16°C. to about 26° C., the first heating cycling temperature is about 66°C. to about 76° C., the second cycling cooling temperature is about 16°C. to about 26° C., the second cycling heating temperature is about 55°C. to about 65° C., the third cycling cooling temperature is about 26°C. to about 36° C., and the third cycling heating temperature is about45° C. to about 55° C. In some embodiments, the first cycling coolingtemperature is about 19° C. to about 23° C., the first heating cyclingtemperature is about 69° C. to about 73° C., the second cycling coolingtemperature is about 19° C. to about 23° C., the second cycling heatingtemperature is about 58° C. to about 62° C., the third cycling coolingtemperature is about 29° C. to about 33° C., and the third cyclingheating temperature is about 48° C. to about 52° C.

In some embodiments, cooling to each cooling temperature is conducted ata substantially different cooling rate. In some embodiments, cooling toeach cooling temperature is conducted at a different cooling rate.

In some embodiments, cooling to the first cycling cooling temperatureand the second cycling cooling temperature are each conducted atsubstantially the same cooling rate.

In some embodiments, the cycling in Step e) comprises the cycling Stepd) three times. In some embodiments, cooling to the first cyclingcooling temperature is conducted at a rate of about 10.0° C./hour toabout 15.0° C./hour. In some embodiments, cooling to the first cyclingcooling temperature is conducted at a rate of about 11.0° C./hour toabout 14.0° C./hour. In some embodiments, cooling to the first cyclingcooling temperature is conducted at a rate of about 12.0° C./hour toabout 13.0° C./hour. In some embodiments, cooling to the first cyclingcooling temperature is conducted at a rate of about 12.5° C./hour.

In some embodiments, the cycling in Step e) comprises the cycling Stepd) three times. In some embodiments, cooling to the second cyclingcooling temperature is conducted at a rate of about 10.0° C./hour toabout 15.0° C./hour. In some embodiments, cooling to the second cyclingcooling temperature is conducted at a rate of about 11.0° C./hour toabout 14.0° C./hour. In some embodiments, cooling to the second cyclingcooling temperature is conducted at a rate of about 12.0° C./hour toabout 13.0° C./hour. In some embodiments, cooling to the first cyclingcooling temperature is conducted at a rate of about 12.5° C./hour.

In some embodiments, the cycling in Step e) comprises the cycling Stepd) three times. In some embodiments, cooling to the third cyclingcooling temperature is conducted at a rate of about 7.5° C./hour toabout 12.5° C./hour. In some embodiments, cooling to the third cyclingcooling temperature is conducted at a rate of about 8.5° C./hour toabout 11.5° C./hour. In some embodiments, cooling to the third cyclingcooling temperature is conducted at a rate of about 9.5° C./hour toabout 10.5° C./hour. In some embodiments, cooling to the second cyclingcooling temperature is conducted at a rate of about 10.0° C./hour.

Four Cycles

In some embodiments, the cycling in Step e) comprises the cycling Stepd) four times.

In some embodiments, cycling Step d) four times comprises: cooling thesuspension to a first cycling cooling temperature, heating thesuspension to a first cycling heating temperature, cooling thesuspension to a second cycling cooling temperature, heating thesuspension to a second cycling heating temperature, cooling thesuspension to a third cycling cooling temperature, heating thesuspension to a third cycling heating temperature, cooling thesuspension to a fourth cycling cooling temperature, and heating thesuspension to a fourth cycling heating temperature.

In some embodiments, the first cycling cooling temperature is about 16°C. to about 26° C., the first heating cycling temperature is about 66°C. to about 76° C., the second cycling cooling temperature is about 16°C. to about 26° C., the second cycling heating temperature is about 66°C. to about 76° C., the third cycling cooling temperature is about 16°C. to about 26° C., the third cycling heating temperature is about 55°C. to about 65° C., the fourth cycling cooling temperature is about 26°C. to about 36° C., and the fourth cycling heating temperature is about45° C. to about 55° C. In some embodiments, the first cycling coolingtemperature is about 19° C. to about 23° C., the first heating cyclingtemperature is about 69° C. to about 73° C., the second cycling coolingtemperature is about 19° C. to about 23° C., the second cycling heatingtemperature is about 69° C. to about 73° C., the third cycling coolingtemperature is about 19° C. to about 23° C., the third cycling heatingtemperature is about 58° C. to about 62° C., the fourth cycling coolingtemperature is about 29° C. to about 33° C., and the fourth cyclingheating temperature is about 48° C. to about 52° C.

In some embodiments, cooling to each cooling temperature is conducted ata substantially different cooling rate. In some embodiments, cooling toeach cooling temperature is conducted at a different cooling rate.

In some embodiments, cooling to the first cycling cooling temperature,the second cycling cooling temperature, and the third cycling coolingtemperature are each conducted at substantially the same cooling rate.

In some embodiments, the cycling in Step e) comprises the cycling Stepd) four times. In some embodiments, cooling to the first cycling coolingtemperature is conducted at a rate of about 10.0° C./hour to about 15.0°C./hour. In some embodiments, cooling to the first cycling coolingtemperature is conducted at a rate of about 11.0° C./hour to about 14.0°C./hour. In some embodiments, cooling to the first cycling coolingtemperature is conducted at a rate of about 12.0° C./hour to about 13.0°C./hour. In some embodiments, cooling to the first cycling coolingtemperature is conducted at a rate of about 12.5° C./hour.

In some embodiments, the cycling in Step e) comprises the cycling Stepd) four times. In some embodiments, cooling to the second cyclingcooling temperature is conducted at a rate of about 10.0° C./hour toabout 15.0° C./hour. In some embodiments, cooling to the second cyclingcooling temperature is conducted at a rate of about 11.0° C./hour toabout 14.0° C./hour. In some embodiments, cooling to the second cyclingcooling temperature is conducted at a rate of about 12.0° C./hour toabout 13.0° C./hour. In some embodiments, cooling to the first cyclingcooling temperature is conducted at a rate of about 12.5° C./hour.

In some embodiments, the cycling in Step e) comprises the cycling Stepd) four times. In some embodiments, cooling to the third cycling coolingtemperature is conducted at a rate of about 10.0° C./hour to about 15.0°C./hour. In some embodiments, cooling to the third cycling coolingtemperature is conducted at a rate of about 11.0° C./hour to about 14.0°C./hour. In some embodiments, cooling to the third cycling coolingtemperature is conducted at a rate of about 12.0° C./hour to about 13.0°C./hour. In some embodiments, cooling to the first cycling coolingtemperature is conducted at a rate of about 12.5° C./hour.

In some embodiments, the cycling in Step e) comprises the cycling Stepd) four times. In some embodiments, cooling to the fourth cyclingcooling temperature is conducted at a rate of about 7.5° C./hour toabout 12.5° C./hour. In some embodiments, cooling to the fourth cyclingcooling temperature is conducted at a rate of about 8.5° C./hour toabout 11.5° C./hour. In some embodiments, cooling to the fourth cyclingcooling temperature is conducted at a rate of about 9.5° C./hour toabout 10.5° C./hour. In some embodiments, cooling to the second cyclingcooling temperature is conducted at a rate of about 10.0° C./hour.

In some embodiments, Step e) is conducted at a stir rate of about 100rpm to about 200 rpm. In some embodiments, Step e) is conducted at astir rate of about 125 rpm to about 175 rpm. In some embodiments, Stepe) is conducted at a stir rate of about 150 rpm.

Step f)—Cooling the Suspension to a Final Cooling Temperature to Formthe Crystalline Free-Plate Habit of L-Arginine Salt of(R)-2-(7-(4-Cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticAcid.

In some embodiments, cooling the suspension in Step f) is conducted at arate of about 7.5° C./hour to about 12.5° C./hour. In some embodiments,cooling the suspension in Step f) is conducted at a rate of about 8.5°C./hour to about 11.5° C./hour. In some embodiments, cooling thesuspension in Step f) is conducted at a rate of about 9.5° C./hour toabout 10.5° C./hour. In some embodiments, cooling the suspension in Stepf) is conducted at a rate of about 10.0° C./hour.

In some embodiments, cooling the suspension in Step f) is conducted at arate of about 8.8° C./hour to about 14.4° C./hour. In some embodiments,cooling the suspension in Step f) is conducted at a rate of about 9.4°C./hour to about 13.8° C./hour. In some embodiments, cooling thesuspension in Step f) is conducted at a rate of about 9.9° C./hour toabout 13.2° C./hour. In some embodiments, cooling the suspension in Stepf) is conducted at a rate of about 10.5° C./hour to about 12.6° C./hour.In some embodiments, cooling the suspension in Step f) is conducted at arate of about 10° C./hour to about 12° C./hour.

In some embodiments, after cooling in Step f) the temperature of thesuspension is about 15° C. to about 40° C. In some embodiments, aftercooling in Step f) the temperature of the suspension is about 20° C. toabout 30° C. In some embodiments, after cooling in Step f) thetemperature of the suspension is about 22° C. to about 24° C. In someembodiments, after cooling in Step f) the temperature of said suspensionis about 18° C. to about 22° C.

In some embodiments, Step f) is conducted at a stir rate of about 100rpm to about 200 rpm. In some embodiments, Step f) is conducted at astir rate of about 125 rpm to about 175 rpm. In some embodiments, Stepf) is conducted at a stir rate of about 150 rpm.

In some embodiments, the method further comprises the step of isolatingthe crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.

In some embodiments, the step of isolating comprises filtering thecrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid from the suspension. In some embodiments, the step of isolatingcomprises filtering the crystalline free-plate habit of L-arginine saltof(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid from the suspension and drying the crystalline free-plate habit ofL-arginine salt at a reduced pressure.

In some embodiments, after filtering, the crystalline free-plate habitof L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has any one or more of the characteristics as described herein.

Another aspect of the present invention relates to pharmaceuticalcompositions prepared according to any of the methods as describedherein.

Another aspect of the present invention relates to a crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid prepared according to any of the methods as described herein.

A composition comprising a crystalline free-plate habit of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid prepared according to any of the methods as described herein.

A pharmaceutical composition comprising a crystalline free-plate habitof L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid prepared according to any of the methods as described herein and apharmaceutical excipient.

In some embodiments, the pharmaceutical composition is suitable fororal, rectal, nasal, topical, buccal, sub-lingual, or vaginal, or in aform suitable for administration by inhalation, insufflation, or by atransdermal patch. In some embodiments, the pharmaceutical compositionis suitable for oral administration.

Crystalline Plate Habit or Morphology

The present invention is directed, inter alia, to a crystallinefree-plate habit or morphology and processes useful in the preparationof a crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)-benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid. The plates were discovered from the novel synthetic methods asdescribed herein and were shown to be thin hexagonal-like plates withtwo opposite sides of the plate being longer that the other sides (i.e.,elongated hexagonal plate). However, due to the thin characteristic ofthe plates, a complete unbroken plate is rarely seen. Instead, what isgenerally observed are large to small broken pieces of the thinhexagonal-like plates. FIG. 5B shows a copy of FIG. 5A that has beenmodified to add an outline to highlight the substantiallyintact/complete free-plate with an elongated hexagonal shape. It isunderstood by those skilled in the art that microscopy is one of themore useful techniques to distinguish two crystalline habits ormorphologies. This is particularly useful when 2 or more morphologiesare associated with the same or substantially the same crystal phase asis the case with the L-arginine salt of Compound 1. Comparing the PXRDpatterns of the habit prepared previously (i.e., WO2011/094008 andExample 2 infra) and the plate habit prepared as described herein (i.e.,see FIG. 19, PXRD overlay between spherulites and plates) it wasobserved that the two PXRD patterns were the same or substantially thesame, thus the two habits represent the same crystal phase. Although thetwo habits revealed the same or substantially the same PXRD pattern, ahigher degree of crystallinity was observed for the plate habit asindicated in FIG. 19 by substantially higher peak intensities and yetlower sample-related background scatter (i.e., a lower amorphous halocontribution). Since sample size and sample preparation can affect peakintensities and sample-related background scatter, and since the twohabits share the same crystal phase, PXRD may not be considered the mostappropriate test method to distinguish between two habits. However, PXRDdoes allow for determining whether two habits have the same crystalphase or different crystal phases. For determining different habits,microscopy is one of the more useful methods. Accordingly, the skilledperson would be capable of reviewing a micrograph for a crystal habitprepared by the processes described herein and readily acknowledge thatthe crystal habit was a free-plate or pieces resulting from a brokenfree-plate and not some other habit, such as, what was preparedpreviously in the art. Similarly, by simple inspection of twomicrographs, such as FIG. 1 (spherulites) and FIG. 5A (free-plates), onecan readily confirm they are different habits.

In addition to the techniques described herein, specific surface canalso be used to characterize the free-plates of the present invention.Accordingly, the specific surface area values disclosed in the presentinvention have been obtained by means of a specific surface areaanalysis technique based on the BET (Brunauer, Emmett and Teller)theory, which is a well-accepted theory known in the art for thecalculation of surface areas of solids by means of measuring theirphysical adsorption of gas molecules (see: Brunauer, S.; Emmett, P. H.;and Teller, E.; J. Am. Chem. Soc., 1938, 60, 309). In particular, thespecific surface area values measured in the present invention have beencalculated from the BET surface area plot obtained by measuring thequantity of nitrogen gas molecules adsorbed by a weighted amount ofsolid at different relative pressures (P/P₀) within the range 0.05-0.3(P/P₀), at 77.3 K. The measurement of the adsorption of gas moleculeswas carried out by means of a Micromeritics™ TriStar II BET surfaceanalyzer having the characteristics as set out below in Example 14.Namely, nitrogen gas was used for the adsorption measurement. The samplefor each analysis was degassed at 25° C. for 960 minutes under vacuum(i.e., 100 mm/Hg). The determination of the adsorption of nitrogen wasmeasured at 77.3 K at eleven relative pressures (P/P₀) sufficientlydispersed within the range of about 0.05 to about 0.30 (i.e. elevenabsolute pressures in the range of about 36 mm Hg to about 223 mm Hgrelative to the saturated pressure at the time of measurement thatranged from about 738 mmHg to about 743 mmHg).

One aspect of the present invention relates to a novel crystalline platemorphology of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid as described herein.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.

In some embodiments, the crystalline free-plate habit has a powder X-raydiffraction pattern comprising peaks, in terms of 2θ, at 8.2°±0.2°,16.4°±0.2°, and 20.5°±0.2°. In some embodiments, the crystallinefree-plate habit has a powder X-ray diffraction pattern comprisingpeaks, in terms of 2θ, at 8.2°±0.2°, 20.5°±0.2°, and 24.6°±0.2°. In someembodiments, the crystalline free-plate habit has a powder X-raydiffraction pattern comprising peaks, in terms of 2θ, at 8.2°±0.2°,16.4°±0.2°, 20.5°±0.2°, and 24.6°±0.2°. In some embodiments, thecrystalline free-plate habit has a powder X-ray diffraction patterncomprising peaks, in terms of 2θ, at 8.2°±0.2°, 16.4°±0.2°, 20.5°±0.2°,24.6°±0.2°, and 28.8°±0.2°. In some embodiments, the crystallinefree-plate habit has a powder X-ray diffraction pattern comprisingpeaks, in terms of 2θ, at 8.2°±0.2°, 16.4°±0.2°, 20.5°±0.2°, 24.6°±0.2°,28.8°±0.2°, and 37.3°±0.2°.

In some embodiments, the crystalline free-plate habit has a differentialscanning calorimetry trace comprising an endotherm with an extrapolatedonset temperature of 205.0° C. to 208.5° C. at a scan rate of 10°C./minute. In some embodiments, the crystalline free-plate habit has adifferential scanning calorimetry trace comprising an endotherm with anextrapolated onset temperature of 205.0° C. to 208.1° C. at a scan rateof 10° C./minute. In some embodiments, the crystalline free-plate habithas a differential scanning calorimetry trace comprising an endothermwith an extrapolated onset temperature of 205.5° C. to 208.5° C. at ascan rate of 10° C./minute. In some embodiments, the crystallinefree-plate habit has a differential scanning calorimetry tracecomprising an endotherm with an extrapolated onset temperature of 206.0°C. to 208.5° C. at a scan rate of 10° C./minute. In some embodiments,the crystalline free-plate habit has a differential scanning calorimetrytrace comprising an endotherm with an extrapolated onset temperature of206.5° C. to 208.5° C. at a scan rate of 10° C./minute. In someembodiments, the crystalline free-plate habit has a differentialscanning calorimetry trace comprising an endotherm with an extrapolatedonset temperature of 205.5° C. to 208.1° C. at a scan rate of 10°C./minute. In some embodiments, the crystalline free-plate habit has adifferential scanning calorimetry trace comprising an endotherm with anextrapolated onset temperature of 206.5° C. to 208.1° C. at a scan rateof 10° C./minute. In some embodiments, the crystalline free-plate habithas a differential scanning calorimetry trace comprising an endothermwith an extrapolated onset temperature of 207.0° C. to 208.1° C. at ascan rate of 10° C./minute. In some embodiments, the crystallinefree-plate habit has a differential scanning calorimetry trace conductedat a scan rate of 10° C./minute comprising an endotherm substantially asdepicted in any one of FIGS. 6 to 10. In some embodiments, thecrystalline free-plate habit has a differential scanning calorimetrytrace conducted at a scan rate of 10° C./minute comprising an endothermsubstantially as depicted in any one of FIGS. 6 to 10 and FIGS. 22 to25.

In some embodiments, the crystalline free-plate habit has a dynamicmoisture sorption (DMS) profile with an adsorption phase from 30% RH to90% RH wherein the crystalline free-plate habit gains about 0.3% weightor less at 90% RH. In some embodiments, the crystalline free-plate habithas a dynamic moisture sorption (DMS) profile with an adsorption phasefrom 30% RH to 90% RH wherein the crystalline free-plate habit gainsabout 0.2% weight or less at 90% RH.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid having a BET specific surface area of about 0.05 m²/g, about 0.1m²/g, about 0.15 m²/g, about 0.2 m²/g, about 0.25 m²/g, about 0.3 m²/g,about 0.35 m²/g, about 0.4 m²/g, about 0.45 m²/g, about 0.5 m²/g, about0.55 m²/g, about 0.6 m²/g, about 0.65 m²/g, or about 0.7 m²/g to about2.0 m²/g, about 2.5 m²/g, about 3.0 m²/g, about 3.5 m²/g, about 4.0m²/g, about 4.5 m²/g, about 5.0 m²/g, about 5.5 m²/g, about 6.0 m²/g,about 6.5 m²/g, about 7.0 m²/g, about 7.5 m²/g, about 8.0 m²/g, about8.5 m²/g, about 9.0 m²/g, or about 9.5 m²/g.

In some embodiments, the crystalline free-plate habit has a BET specificsurface area of about 0.1 m²/g to about 5.0 m²/g. In some embodiments,the crystalline free-plate habit has a BET specific surface area ofabout 0.1 m²/g to about 4.0 m²/g. In some embodiments, the crystallinefree-plate habit has a BET specific surface area of about 0.3 m²/g toabout 4.0 m²/g. In some embodiments, the crystalline free-plate habithas a BET specific surface area of about 0.5 m²/g to about 4.0 m²/g. Insome embodiments, the crystalline free-plate habit has a BET specificsurface area of about 0.6 m²/g to about 4.0 m²/g. In some embodiments,the crystalline free-plate habit has a BET specific surface area ofabout 0.3 m²/g to about 3.0 m²/g. In some embodiments, the crystallinefree-plate habit has a BET specific surface area of about 0.4 m²/g toabout 2.0 m²/g. In some embodiments, the crystalline free-plate habithas a BET specific surface area of about 0.5 m²/g to about 1.8 m²/g. Insome embodiments, the crystalline free-plate habit has a BET specificsurface area of about 0.6 m²/g to about 1.6 m²/g.

In some embodiments, the crystalline free-plate habit has:

1) a powder X-ray diffraction pattern comprising peaks, in terms of 2θ,at 8.2°±0.2°, 16.40°±0.2°, 20.5°±0.2°, 24.6°±0.2°, 28.8°±0.2°, and37.3°±0.2°;

2) a differential scanning calorimetry trace comprising an endothermwith an extrapolated onset temperature of 205.0° C. to 208.5° C. at ascan rate of 10° C./minute; and/or

3) a dynamic moisture sorption (DMS) profile with an adsorption phasefrom 30% RH to 90% RH wherein the crystalline free-plate habit gainsabout 0.3% weight or less at 90% RH.

In some embodiments, the crystalline free-plate habit has:

1) a powder X-ray diffraction pattern comprising peaks, in terms of 2θ,at 8.2°±0.2°, 16.40°±0.2°, 20.5°±0.2°, and 24.6°±0.2°;

2) a differential scanning calorimetry trace comprising an endothermwith an extrapolated onset temperature of 206.5° C. to t 208.5° C. at ascan rate of 10° C./minute; and/or

3) a dynamic moisture sorption (DMS) profile with an adsorption phasefrom 30% RH to 90% RH wherein the crystalline free-plate habit gainsabout 0.3% weight or less at 90% RH.

In some embodiments, the crystalline free-plate habit has:

1) a powder X-ray diffraction pattern comprising peaks, in terms of 2θ,at 8.2°±0.2°, 20.50°±0.2°, and 24.6°±0.2°;

2) a differential scanning calorimetry trace comprising an endothermwith an extrapolated onset temperature of 205.5° C. to 208.5° C. at ascan rate of 10° C./minute; and/or

3) a dynamic moisture sorption (DMS) profile with an adsorption phasefrom 30% RH to 90% RH wherein the crystalline free-plate habit gainsabout 0.2% weight or less at 90% RH.

In some embodiments, the crystalline free-plate habit has:

1) a powder X-ray diffraction pattern comprising peaks, in terms of 2θ,at 8.2°±0.2°, 16.40°±0.2°, and 20.5°±0.2°;

2) a differential scanning calorimetry trace comprising an endothermwith an extrapolated onset temperature of 207.1° C. to 208.1° C. at ascan rate of 10° C./minute; and/or

3) a dynamic moisture sorption (DMS) profile with an adsorption phasefrom 30% RH to 90% RH wherein the crystalline free-plate habit gainsabout 0.2% weight or less at 90% RH.

In some embodiments, the crystalline free-plate habit has:

1) a differential scanning calorimetry trace comprising an endothermwith an extrapolated onset temperature of 205.0° C. to 208.5° C. at ascan rate of 10° C./minute; and/or

2) a dynamic moisture sorption (DMS) profile with an adsorption phasefrom 30% RH to 90% RH wherein the crystalline free-plate habit gainsabout 0.3% weight or less at 90% RH.

In some embodiments, the crystalline free-plate habit has:

1) a differential scanning calorimetry trace comprising an endothermwith an extrapolated onset temperature of 206.5° C. to t 208.5° C. at ascan rate of 10° C./minute; and/or

2) a dynamic moisture sorption (DMS) profile with an adsorption phasefrom 30% RH to 90% RH wherein the crystalline free-plate habit gainsabout 0.3% weight or less at 90% RH.

In some embodiments, the crystalline free-plate habit has:

1) a differential scanning calorimetry trace comprising an endothermwith an extrapolated onset temperature of 205.5° C. to 208.5° C. at ascan rate of 10° C./minute; and/or

2) a dynamic moisture sorption (DMS) profile with an adsorption phasefrom 30% RH to 90% RH wherein the crystalline free-plate habit gainsabout 0.2% weight or less at 90% RH.

In some embodiments, the crystalline free-plate habit has:

1) a differential scanning calorimetry trace comprising an endothermwith an extrapolated onset temperature of 207.1° C. to 208.1° C. at ascan rate of 10° C./minute; and/or

2) a dynamic moisture sorption (DMS) profile with an adsorption phasefrom 30% RH to 90% RH wherein the crystalline free-plate habit gainsabout 0.2% weight or less at 90% RH. In some embodiments, thecrystalline free-plate habit has:

1) a powder X-ray diffraction pattern comprising peaks, in terms of 2θ,at 8.2°±0.2°, 16.40°±0.2°, and 20.5°±0.2°;

2) a differential scanning calorimetry trace comprising an endothermwith an extrapolated onset temperature of 205.0° C. to 208.5° C. at ascan rate of 10° C./minute;

3) a dynamic moisture sorption (DMS) profile with an adsorption phasefrom 30% RH to 90% RH wherein said crystalline free-plate habit gainsabout 0.3% weight or less at 90% RH; and/or

4) a BET specific surface area of about 0.1 m²/g to about 5.0 m²/g. Insome embodiments, the crystalline free-plate habit has:

1) a powder X-ray diffraction pattern comprising peaks, in terms of 2θ,at 8.2°±0.2°, 20.50°±0.2°, and 24.6°±0.2°;

2) a differential scanning calorimetry trace comprising an endothermwith an extrapolated onset temperature of 205.5° C. to t 208.5° C. at ascan rate of 10° C./minute;

3) a dynamic moisture sorption (DMS) profile with an adsorption phasefrom 30% RH to 90% RH wherein said crystalline free-plate habit gainsabout 0.3% weight or less at 90% RH; and/or

4) a BET specific surface area of about 0.1 m²/g to about 4.0 m²/g.

In some embodiments, the crystalline free-plate habit has:

1) a powder X-ray diffraction pattern comprising peaks, in terms of 2θ,at 8.2°±0.2°, 20.50°±0.2°, and 24.6°±0.2°;

2) a differential scanning calorimetry trace comprising an endothermwith an extrapolated onset temperature of 205.5° C. to t 208.5° C. at ascan rate of 10° C./minute;

3) a dynamic moisture sorption (DMS) profile with an adsorption phasefrom 30% RH to 90% RH wherein said crystalline free-plate habit gainsabout 0.3% weight or less at 90% RH; and/or

4) a BET specific surface area of about 0.3 m²/g to about 3.0 m²/g.

In some embodiments, the crystalline free-plate habit has:

1) a powder X-ray diffraction pattern comprising peaks, in terms of 2θ,at 8.2°±0.2°, 16.40°±0.2°, 20.5°±0.2°, and 24.6°±0.2°;

2) a differential scanning calorimetry trace comprising an endothermwith an extrapolated onset temperature of 205.5° C. to 208.5° C. at ascan rate of 10° C./minute;

3) a dynamic moisture sorption (DMS) profile with an adsorption phasefrom 30% RH to 90% RH wherein said crystalline free-plate habit gainsabout 0.3% weight or less at 90% RH; and/or

4) a BET specific surface area of about 0.6 m²/g to about 4.0 m²/g.

In some embodiments, the crystalline free-plate habit has:

1) a powder X-ray diffraction pattern comprising peaks, in terms of 2θ,at 8.2°±0.2°, 16.40°±0.2°, 20.5°±0.2°, and 24.6°±0.2°;

2) a differential scanning calorimetry trace comprising an endothermwith an extrapolated onset temperature of 206.5° C. to t 208.5° C. at ascan rate of 10° C./minute;

3) a dynamic moisture sorption (DMS) profile with an adsorption phasefrom 30% RH to 90% RH wherein said crystalline free-plate habit gainsabout 0.3% weight or less at 90% RH; and/or

4) a BET specific surface area of about 0.4 m²/g to about 2.0 m²/g.

In some embodiments, the crystalline free-plate habit has:

1) a powder X-ray diffraction pattern comprising peaks, in terms of 2θ,at 8.2°±0.2°, 16.40°±0.2°, 20.5°±0.2°, 24.6°±0.2°, and 28.8°±0.2°;

2) a differential scanning calorimetry trace comprising an endothermwith an extrapolated onset temperature of 206.5° C. to 208.1° C. at ascan rate of 10° C./minute;

3) a dynamic moisture sorption (DMS) profile with an adsorption phasefrom 30% RH to 90% RH wherein said crystalline free-plate habit gainsabout 0.2% weight or less at 90% RH; and/or

4) a BET specific surface area of about 0.5 m²/g to about 1.8 m²/g.

In some embodiments, the crystalline free-plate habit has:

1) a powder X-ray diffraction pattern comprising peaks, in terms of 2θ,at 8.2°±0.2°, 16.40°±0.2°, 20.5°±0.2°, 24.6°±0.2°, 28.8°±0.2°, and37.3°±0.2°;

2) a differential scanning calorimetry trace comprising an endothermwith an extrapolated onset temperature of 205.5° C. to 208.1° C. at ascan rate of 10° C./minute;

3) a dynamic moisture sorption (DMS) profile with an adsorption phasefrom 30% RH to 90% RH wherein said crystalline free-plate habit gainsabout 0.2% weight or less at 90% RH; and/or

4) a BET specific surface area of about 0.6 m2/g to about 4.0 m2/g.

In some embodiments, the crystalline free-plate habit has:

1) a powder X-ray diffraction pattern comprising peaks, in terms of 2θ,at 8.2°±0.2°, 16.40°±0.2°, 20.5°±0.2°, 24.6°±0.2°, 28.8°±0.2°, and37.3°±0.2°;

2) a differential scanning calorimetry trace comprising an endothermwith an extrapolated onset temperature of 207.1° C. to 208.1° C. at ascan rate of 10° C./minute;

3) a dynamic moisture sorption (DMS) profile with an adsorption phasefrom 30% RH to 90% RH wherein said crystalline free-plate habit gainsabout 0.2% weight or less at 90% RH; and/or

4) a BET specific surface area of about 0.6 m²/g to about 1.6 m²/g.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid having a powder X-ray diffraction pattern comprising peaks, interms of 2θ, at 8.2°±0.2°, 16.40°±0.2°, and 20.5°±0.2°. In someembodiments, the crystalline free-plate habit has a powder X-raydiffraction pattern comprising peaks, in terms of 2θ, at 8.2°±0.2°,20.5°±0.2°, and 24.6°±0.2°. In some embodiments, the crystallinefree-plate habit has a powder X-ray diffraction pattern comprisingpeaks, in terms of 2θ, at 8.2°±0.2°, 16.4°±0.2°, 20.5°±0.2°, and24.6°±0.2°. In some embodiments, the crystalline free-plate habit has apowder X-ray diffraction pattern comprising peaks, in terms of 2θ, at8.2°±0.2°, 16.4°±0.2°, 20.5°±0.2°, 24.6°±0.2°, and 28.8°±0.2°. In someembodiments, the crystalline free-plate habit has a powder X-raydiffraction pattern comprising peaks, in terms of 2θ, at 8.2°±0.2°,16.4°±0.2°, 20.5°±0.2°, 24.6°±0.2°, 28.8°±0.2°, and 37.3°±0.2°.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid having a differential scanning calorimetry trace comprising anendotherm with an extrapolated onset temperature of 205.0° C. to 208.5°C. when scanned at 10° C. per minute. In some embodiments, thecrystalline free-plate habit has a differential scanning calorimetrytrace comprising an endotherm with an extrapolated onset temperature of205.0° C. to 208.1° C. at a scan rate of 10° C./minute. In someembodiments, the crystalline free-plate habit has a differentialscanning calorimetry trace comprising an endotherm with an extrapolatedonset temperature of 205.5° C. to 208.5° C. at a scan rate of 10°C./minute. In some embodiments, the crystalline free-plate habit has adifferential scanning calorimetry trace comprising an endotherm with anextrapolated onset temperature of 205.5° C. to 208.1° C. at a scan rateof 10° C./minute. In some embodiments, the crystalline free-plate habithas a differential scanning calorimetry trace comprising an endothermwith an extrapolated onset temperature of 206.0° C. to 208.5° C. at ascan rate of 10° C./minute. In some embodiments, the crystallinefree-plate habit has a differential scanning calorimetry tracecomprising an endotherm with an extrapolated onset temperature of 206.5°C. to 208.5° C. at a scan rate of 10° C./minute. In some embodiments,the crystalline free-plate habit has a differential scanning calorimetrytrace comprising an endotherm with an extrapolated onset temperature of206.5° C. to 208.1° C. at a scan rate of 10° C./minute. In someembodiments, the crystalline free-plate habit has a differentialscanning calorimetry trace comprising an endotherm with an extrapolatedonset temperature of 207.0° C. to 208.1° C. at a scan rate of 10°C./minute. In some embodiments, the crystalline free-plate habit has adifferential scanning calorimetry trace conducted at a scan rate of 10°C./minute comprising an endotherm substantially as depicted in any oneof FIGS. 6 to 10. In some embodiments, the crystalline free-plate habithas a differential scanning calorimetry trace conducted at a scan rateof 10° C./minute comprising an endotherm substantially as depicted inany one of FIGS. 6 to 10 and FIGS. 22 to 25. In some embodiments, thecrystalline free-plate habit has a powder X-ray diffraction patterncomprising peaks, in terms of 2θ, at 8.2°±0.2°, 16.4°±0.2°, and20.5°±0.2°. In some embodiments, the crystalline free-plate habit has apowder X-ray diffraction pattern comprising peaks, in terms of 2θ, at8.2°±0.2°, 20.5°±0.2°, and 24.6°±0.2°. In some embodiments, thecrystalline free-plate habit has a powder X-ray diffraction patterncomprising peaks, in terms of 2θ, at 8.2°±0.2°, 16.4°±0.2°, 20.5°±0.2°,and 24.6°±0.2°. In some embodiments, the crystalline free-plate habithas a powder X-ray diffraction pattern comprising peaks, in terms of 2θ,at 8.2°±0.2°, 16.4°±0.2°, 20.5°±0.2°, 24.6°±0.2°, and 28.8°±0.2°. Insome embodiments, the crystalline free-plate habit has a powder X-raydiffraction pattern comprising peaks, in terms of 2θ, at 8.2°±0.2°,16.4°±0.2°, 20.5°±0.2°, 24.6°±0.2°, 28.8°±0.2°, and 37.3°±0.2°.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid having a dynamic moisture sorption (DMS) profile with an adsorptionphase from 30% RH to 90% RH wherein the crystalline free-plate habitgains about 0.3% weight or less at 90% RH. In some embodiments, thecrystalline free-plate habit has a dynamic moisture sorption (DMS)profile with an adsorption phase from 30% RH to 90% RH wherein thecrystalline free-plate habit gains about 0.2% weight or less at 90% RH.In some embodiments, the crystalline free-plate habit has a powder X-raydiffraction pattern comprising peaks, in terms of 2θ, at 8.2°±0.2°,16.4°±0.2°, and 20.5°±0.2°. In some embodiments, the crystallinefree-plate habit has a powder X-ray diffraction pattern comprisingpeaks, in terms of 2θ, at 8.2°±0.2°, 20.5°±0.2°, and 24.6°±0.2°. In someembodiments, the crystalline free-plate habit has a powder X-raydiffraction pattern comprising peaks, in terms of 2θ, at 8.2°±0.2°,16.4°±0.2°, 20.5°±0.2°, and 24.6°±0.2°. In some embodiments, thecrystalline free-plate habit has a powder X-ray diffraction patterncomprising peaks, in terms of 2θ, at 8.2°±0.2°, 16.40°±0.2°, 20.5°±0.2°,24.6°±0.2°, and 28.8°±0.2°. In some embodiments, the crystallinefree-plate habit has a powder X-ray diffraction pattern comprisingpeaks, in terms of 2θ, at 8.2°±0.2°, 16.4°±0.2°, 20.5°±0.2°, 24.6°±0.2°,28.8°±0.2°, and 37.3°±0.2°.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid having a BET specific surface area of about 0.1 m²/g to about 5.0m²/g. In some embodiments, the crystalline free-plate habit has a BETspecific surface area of about 0.1 m²/g to about 4.0 m²/g. In someembodiments, the crystalline free-plate habit has a BET specific surfacearea of about 0.3 m²/g to about 4.0 m²/g. In some embodiments, thecrystalline free-plate habit has a BET specific surface area of about0.5 m²/g to about 4.0 m²/g. In some embodiments, the crystallinefree-plate habit has a BET specific surface area of about 0.6 m²/g toabout 4.0 m²/g. In some embodiments, the crystalline free-plate habithas a BET specific surface area of about 0.3 m²/g to about 3.0 m²/g. Insome embodiments, the crystalline free-plate habit has a BET specificsurface area of about 0.4 m²/g to about 2.0 m²/g. In some embodiments,the crystalline free-plate habit has a BET specific surface area ofabout 0.5 m²/g to about 1.8 m²/g. In some embodiments, the crystallinefree-plate habit has a BET specific surface area of about 0.6 m²/g toabout 1.6 m²/g. In some embodiments, the crystalline free-plate habithas a powder X-ray diffraction pattern comprising peaks, in terms of 2θ,at 8.2°±0.2°, 16.4°±0.2°, and 20.5°±0.2°. In some embodiments, thecrystalline free-plate habit has a powder X-ray diffraction patterncomprising peaks, in terms of 2θ, at 8.2°±0.2°, 20.5°±0.2°, and24.6°±0.2°. In some embodiments, the crystalline free-plate habit has apowder X-ray diffraction pattern comprising peaks, in terms of 2θ, at8.2°±0.2°, 16.4°±0.2°, 20.5°±0.2°, and 24.6°±0.2°. In some embodiments,the crystalline free-plate habit has a powder X-ray diffraction patterncomprising peaks, in terms of 2θ, at 8.2°±0.2°, 16.40°±0.2°, 20.5°±0.2°,24.6°±0.2°, and 28.8°±0.2°. In some embodiments, the crystallinefree-plate habit has a powder X-ray diffraction pattern comprisingpeaks, in terms of 2θ, at 8.2°±0.2°, 16.4°±0.2°, 20.5°±0.2°, 24.6°±0.2°,28.8°±0.2°, and 37.3°±0.2°.

Indications

S1P receptor agonists having agonist activity on the S1P₁ receptor havebeen shown to rapidly and reversibly induce lymphopenia (also referredto as peripheral lymphocyte lowering (PLL); Hale et al., Bioorg. MedChem. Lett., 14:3351-3355, 2004). This is attended by clinically usefulimmunosuppression by virtue of sequestering T- and B-cells in secondarylymphoid tissue (lymph nodes and Peyer's patches) and thus apart fromsites of inflammation and organ grafts (Rosen et al., Immunol. Rev.,195:160-177, 2003; Schwab et al., Nature Immunol., 8:1295-1301, 2007).This lymphocyte sequestration, for example in lymph nodes, is thought tobe a consequence of concurrent agonist-driven functional antagonism ofthe S1P₁ receptor on T-cells (whereby the ability of S1P to mobilizeT-cell egress from lymph nodes is reduced) and persistent agonism of theS1P₁ receptor on lymph node endothelium (such that barrier functionopposing transmigration of lymphocytes is increased) (Matloubian et al.,Nature, 427:355-360, 2004; Baumruker et al., Expert Opin. Investig.Drugs, 16:283-289, 2007). It has been reported that agonism of the S1P₁receptor alone is sufficient to achieve lymphocyte sequestration (Sannaet al., J Biol Chem., 279:13839-13848, 2004) and that this occurswithout impairment of immune responses to systemic infection (Brinkmannet al., Transplantation, 72:764-769, 2001; Brinkmann et al., TransplantProc., 33:530-531, 2001).

That agonism of endothelial S1P₁ receptors has a broader role inpromoting vascular integrity is supported by work implicating the S1P₁receptor in capillary integrity in mouse skin and lung (Sanna et al.,Nat Chem Biol., 2:434-441, 2006). Vascular integrity can be compromisedby inflammatory processes, for example as may derive from sepsis, majortrauma and surgery so as to lead to acute lung injury or respiratorydistress syndrome (Johan Groeneveld, Vascul. Pharmacol., 39:247-256,2003).

An exemplary S1P receptor agonist having agonist activity on the S1P₁receptor is FTY720, an immunosuppressive agent (Martini et al., ExpertOpin. Investig. Drugs, 16:505-518, 2007) that was approved by the FDA asGilenya® (fingolimod) in September 2010 for the treatment of relapsingmultiple sclerosis. FTY720 acts as a prodrug which is phosphorylated invivo; the phosphorylated derivative is an agonist for S1P₁, S1P₃, S1P₄,and S1P₅ receptors (but not the S1P₂ receptor) (Chiba, Pharmacology &Therapeutics, 108:308-319, 2005). FTY720 has been shown to rapidly andreversibly induce lymphopenia (also referred to as peripheral lymphocytelowering (PLL); Hale et al., Bioorg. Med. Chem. Lett., 14:3351-3355,2004). This is attended by clinically useful immunosuppression by virtueof sequestering T- and B-cells in secondary lymphoid tissue (lymph nodesand Peyer's patches) and thus apart from sites of inflammation and organgrafts (Rosen et al., Immunol. Rev., 195:160-177, 2003; Schwab et al.,Nature Immunol., 8:1295-1301, 2007).

FTY720 has been reported to have therapeutic efficacy in at least: a ratmodel for autoimmune myocarditis and a mouse model for acute viralmyocarditis (Kiyabayashi et al., J. Cardiovasc. Pharmacol., 35:410-416,2000; Miyamoto et al., J. Am. Coll. Cardiol., 37:1713-1718, 2001); mousemodels for inflammatory bowel disease including colitis (Mizushima etal., Inflamm. Bowel Dis., 10:182-192, 2004; Deguchi et al., OncologyReports, 16:699-703, 2006; Fujii et al., Am. J. Physiol. Gastrointest.Liver Physiol., 291:G267-G274, 2006; Daniel et al., J. Immunol.,178:2458-2468, 2007); a rat model for progressive mesangioproliferativeglomerulonephritis (Martini et al., Am. J. Physiol. Renal Physiol.,292:F1761-F1770, 2007); a mouse model for asthma, suggested to beprimarily through the S1P₁ receptor on the basis of work using the S1P₁receptor agonist SEW2871 (Idzko et al, J. Clin. Invest., 116:2935-2944,2006); a mouse model for airway inflammation and induction of bronchialhyperresponsiveness (Sawicka et al., J. Immunol., 171; 6206-6214, 2003);a mouse model for atopic dermatitis (Kohno et al., Biol. Pharm. Bull.,27:1392-1396, 2004); a mouse model for ischemia-reperfusion injury(Kaudel et al., Transplant. Proc, 39:499-502, 2007); a mouse model forsystemic lupus erythematosus (SLE) (Okazaki et al., J. Rheumatol.,29:707-716, 2002; Herzinger et al, Am. J. Clin. Dermatol., 8:329-336,2007); rat models for rheumatoid arthritis (Matsuura et al., Int. J.Immunopharmacol., 22:323-331, 2000; Matsuura et al., Inflamm. Res.,49:404-410, 2000); a rat model for autoimmune uveitis (Kurose et al.,Exp. Eye Res., 70:7-15, 2000); mouse models for type I diabetes (Fu etal, Transplantation, 73:1425-1430, 2002; Maki et al., Transplantation,74:1684-1686, 2002; Yang et al., Clinical Immunology, 107:30-35, 2003;Maki et al., Transplantation, 79:1051-1055, 2005); mouse models foratherosclerosis (Nofer et al., Circulation, 115:501-508, 2007; Keul etal., Arterioscler. Thromb. Vasc. Biol., 27:607-613, 2007); a rat modelfor brain inflammatory reaction following traumatic brain injury (TBI)(Zhang et al., J. Cell. Mol. Med., 11:307-314, 2007); and mouse modelsfor graft coronary artery disease and graft-versus-host disease (GVHD)(Hwang et al., Circulation, 100: 1322-1329, 1999; Taylor et al., Blood,110:3480-3488, 2007). In vitro results suggest that FTY720 may havetherapeutic efficacy for β-amyloid-related inflammatory diseasesincluding Alzheimer's disease (Kaneider et al., FASEB J., 18:309-311,2004). KRP-203, an S1P receptor agonist having agonist activity on theS1P₁ receptor, has been reported to have therapeutic efficacy in a ratmodel for autoimmune myocarditis (Ogawa et al., BBRC, 361:621-628,2007). Using the S1P₁ receptor agonist SEW2871, it has been shown thatagonism of endothelial S1P₁ receptors prevents proinflammatorymonocyte/endothelial interactions in type I diabetic vascularendothelium (Whetzel et al., Circ. Res., 99:731-739, 2006) and protectsthe vasculature against TNFα-mediated monocyte/endothelial interactions(Bolick et al., Arterioscler. Thromb. Vasc. Biol., 25:976-981, 2005).

Additionally, FTY720 has been reported to have therapeutic efficacy inexperimental autoimmune encephalomyelitis (EAE) in rats and mice, amodel for human multiple sclerosis (Brinkmann et al., J. Biol. Chem.,277:21453-21457, 2002; Fujino et al., J. Pharmacol. Exp. Ther.,305:70-77, 2003; Webb et al., J. Neuroimmunol., 153:108-121, 2004;Rausch et al., J. Magn. Reson. Imaging, 20:16-24, 2004; Kataoka et al.,Cellular & Molecular Immunology, 2:439-448, 2005; Brinkmann et al.,Pharmacology & Therapeutics, 115:84-105, 2007; Baumruker et al., ExpertOpin. Investig. Drugs, 16:283-289, 2007; Balatoni et al., Brain ResearchBulletin, 74:307-316, 2007). Furthermore, FTY720 has been found to havetherapeutic efficacy for multiple sclerosis in clinical trials. In PhaseII clinical trials for relapsing-remitting multiple sclerosis, FTY720was found to reduce the number of lesions detected by magnetic resonanceimaging (MRI) and clinical disease activity in patients with multiplesclerosis (Kappos et al., N. Engl. J. Med., 355:1124-1140, 2006; Martiniet al., Expert Opin. Investig. Drugs, 16:505-518, 2007; Zhang et al.,Mini-Reviews in Medicinal Chemistry, 7:845-850, 2007; Brinkmann,Pharmacology & Therapeutics, 115:84-105, 2007). FTY720 is currently inPhase III studies of remitting-relapsing multiple sclerosis (Brinkmann,Pharmacology & Therapeutics, 115:84-105, 2007; Baumruker et al., Expert.Opin. Investig. Drugs, 16:283-289, 2007; Dev et al., Pharmacology andTherapeutics, 117:77-93, 2008).

FTY720 has also been reported to have anti-viral activity. Specific datahas been presented in the lymphocytic choriomeningitis virus (LCMV)mouse model, wherein the mice were infected with either the Armstrong orthe clone 13 strain of LCMV (Premenko-Lanier et al., Nature, 454, 894,2008).

FTY720 has also been reported to impair migration of dendritic cellsinfected with Francisella tularensis to the mediastinal lymph node,thereby reducing the bacterial colonization of it. Francisellatularensis is associated with tularemia, ulceroglandular infection,respiratory infection and a typhoidal disease (E. Bar-Haim et al., PLoSPathog 4(11): e1000211. doi:10.1371/journal.ppat. 1000211, 2008).

It has also been reported that a short-term high dose of FTY720 rapidlyreduced ocular infiltrates in experimental autoimmune uveoretinitis.When given in the early stages of ocular inflammation, FTY720 rapidlyprevented retinal damage. It was reported to not only preventinfiltration of target organs, but also reduce existing infiltration(Raveney et al., Arch. Ophthalmol. 126(10), 1390, 2008).

It has been reported that treatment with FTY720 relievedovariectomy-induced osteoporosis in mice by reducing the number ofmature osteoclasts attached to the bone surface. The data providedevidence that SIP controlled the migratory behavior of osteoclastprecursors, dynamically regulating bone mineral homeostasis (Ishii etal., Nature, 458(7237), 524-528, 2009).

Agonism of the S1P₁ receptor has been implicated in enhancement ofsurvival of oligodendrocyte progenitor cells. Survival ofoligodendrocyte progenitor cells is a required component of theremyelination process. Remyelination of multiple sclerosis lesions isconsidered to promote recovery from clinical relapses. (Miron et al.,Ann. Neurol., 63:61-71, 2008; Coelho et al., J. Pharmacol. Exp. Ther.,323:626-635, 2007; Dev et al., Pharmacology and Therapeutics, 117:77-93,2008). It also has been shown that the S1P₁ receptor plays a role inplatelet-derived growth factor (PDGF)-induced oligodendrocyte progenitorcell mitogenesis (Jung et al., Glia, 55:1656-1667, 2007).

Agonism of the S1P₁ receptor has also been reported to mediate migrationof neural stem cells toward injured areas of the central nervous system(CNS), including in a rat model of spinal cord injury (Kimura et al.,Stem Cells, 25:115-124, 2007).

Agonism of the S1P₁ receptor has been implicated in the inhibition ofkeratinocyte proliferation (Sauer et al., J. Biol. Chem.,279:38471-38479, 2004), consistent with reports that S1P inhibitskeratinocyte proliferation (Kim et al., Cell Signal, 16:89-95, 2004).The hyperproliferation of keratinocytes at the entrance to the hairfollicle, which can then become blocked, and an associated inflammationare significant pathogenetic factors of acne (Koreck et al.,Dermatology, 206:96-105, 2003; Webster, Cutis, 76:4-7, 2005).

FTY720 has been reported to have therapeutic efficacy in inhibitingpathologic angiogenesis, such as that as may occur in tumor development.Inhibition of angiogenesis by FTY720 is thought to involve agonism ofthe S1P₁ receptor (Oo et al., J. Biol. Chem., 282; 9082-9089, 2007;Schmid et al., J. Cell Biochem., 101:259-270, 2007). FTY720 has beenreported to have therapeutic efficacy for inhibiting primary andmetastatic tumor growth in a mouse model of melanoma (LaMontagne et al.,Cancer Res., 66:221-231, 2006). FTY720 has been reported to havetherapeutic efficacy in a mouse model for metastatic hepatocellularcarcinoma (Lee et al., Clin. Cancer Res., 11:84588466, 2005).

It has been reported that oral administration of FTY720 to mice potentlyblocked VEGF-induced vascular permeability, an important processassociated with angiogenesis, inflammation, and pathological conditionssuch as sepsis, hypoxia, and solid tumor growth (T Sanchez et al., J.Biol. Chem., 278(47), 47281-47290, 2003).

FTY720 has been shown to have therapeutic efficacy in transplantrejection both as a monotherapy and in synergistic combination with aclassical immunosuppressant, including cyclosporin A, FK506 and RAD (anmTOR inhibitor). It has been shown that, unlike the classicalimmunosuppressants cyclosporin A, FK506 and RAD, FTY720 has efficacy forprolonging graft survival without inducing general immunosuppression,and this difference in drug action is believed to be relevant to thesynergism observed for the combination (Brinkmann et al., TransplantProc., 33:530-531, 2001; Brinkmann et al., Transplantation, 72:764-769,2001).

Agonism of the S1P₁ receptor has been reported to have therapeuticefficacy for prolonging allograft survival in mouse and rat skinallograft models (Lima et al., Transplant Proc., 36:1015-1017, 2004; Yanet al., Bioorg. & Med. Chem. Lett., 16:3679-3683, 2006). FTY720 has beenreported to have therapeutic efficacy for prolonging allograft survivalin a rat cardiac allograft model (Suzuki et al., Transpl. Immunol.,4:252-255, 1996). FTY720 has been reported to act synergistically withcyclosporin A to prolong rat skin allograft survival (Yanagawa et al.,J. Immunol., 160:5493-5499, 1998), to act synergistically withcyclosporin A and with FK506 to prolong rat cardiac allograft survival,and to act synergistically with cyclosporin A to prolong canine renalallograft survival and monkey renal allograft survival (Chiba et al.,Cell Mol. Biol., 3:11-19, 2006). KRP-203, an S1P receptor agonist hasbeen reported to have therapeutic efficacy for prolonging allograftsurvival in a rat skin allograft model and both as monotherapy and insynergistic combination with cyclosporin A in a rat cardiac allograftmodel (Shimizu et al., Circulation, 111:222-229, 2005). KRP-203 also hasbeen reported to have therapeutic efficacy in combination withmycophenolate mofetil (MMF; a prodrug for which the active metabolite ismycophenolic acid, an inhibitor of purine biosynthesis) for prolongingallograft survival both in a rat renal allograft model and in a ratcardiac allograft model (Suzuki et al., J. Heart Lung Transplant,25:302-209, 2006; Fujishiro et al., J. Heart Lung Transplant,25:825-833, 2006). It has been reported that an agonist of the S1P₁receptor, AUY954, in combination with a subtherapeutic dose of RAD001(Certican/Everolimus, an mTOR inhibitor) can prolong rat cardiacallograft survival (Pan et al., Chemistry & Biology, 13:1227-1234,2006). In a rat small bowel allograft model, FTY720 has been reported toact synergistically with cyclosporin A to prolong small bowel allograftsurvival (Sakagawa et al., Transpl. Immunol., 13:161-168, 2004). FTY720has been reported to have therapeutic efficacy in a mouse islet graftmodel (Fu et al., Transplantation, 73:1425-1430, 2002; Liu et al.,Microsurgery, 27:300-304; 2007) and in a study using human islet cellsto evidence no detrimental effects on human islet function (Truong etal., American Journal of Transplantation, 7:2031-2038, 2007).

FTY720 has been reported to reduce the nociceptive behavior in thespared nerve injury model for neuropathic pain which does not depend onprostaglandin synthesis (O. Costu et al., Journal of Cellular andMolecular Medicine 12(3), 995-1004, 2008).

FTY720 has been reported to impair initiation of murine contacthypersensitivity (CHS). Adoptive transfer of immunized lymph node cellsfrom mice treated with FTY720 during the sensitization phase wasvirtually incapable of inducing CHS response in recipients (D. Nakashimaet al., J. Investigative Dermatology (128(12), 2833-2841, 2008).

It has been reported that prophylactic oral administration of FTY720 (1mg/kg, three times a week), completely prevented the development ofexperimental autoimmune myasthenia gravis (EAMG) in C57BL/6 mice (T.Kohono et al., Biological & Pharmaceutical Bulletin, 28(4), 736-739,2005).

S1P₁ receptor agonists are useful for treating or preventing conditionswhere suppression of the immune system or agonism of the S1P₁ receptoris in order, such as diseases and disorders mediated by lymphocytes,transplant rejection, autoimmune diseases and disorders, inflammatorydiseases and disorders, and conditions that have an underlying defect invascular integrity or that relate to angiogenesis such as may bepathologic.

In some embodiments, an S1P₁ receptor-associated disorder is selectedfrom, for example, diseases and disorders mediated by lymphocytes,transplant rejection, autoimmune diseases and disorders, inflammatorydiseases and disorders (e.g., acute and chronic inflammatoryconditions), cancer, and conditions that have an underlying defect invascular integrity or that are associated with angiogenesis such as maybe pathologic (e.g., as may occur in inflammation, tumor development andatherosclerosis). Such conditions where suppression of the immune systemor agonism of the S1P₁ receptor is in order include diseases anddisorders mediated by lymphocytes, conditions that have an underlyingdefect in vascular integrity, autoimmune diseases and disorders,inflammatory diseases and disorders (e.g., acute and chronicinflammatory conditions), acute or chronic rejection of cells, anautoimmune disease of the liver including biliary cirrhosis, primarybiliary cirrhosis, autoimmune hepatitis, primary sclerosing cholangitis,and primary biliary cholangitis, tissue or solid organ grafts, arthritisincluding psoriatic arthritis and rheumatoid arthritis, diabetesincluding type I diabetes and the disorders associated therewith,myasthenia gravis, demyelinating disease including multiple sclerosis,ischemia-reperfusion injury including renal and cardiacischemia-reperfusion injury, inflammatory skin disease includingpsoriasis, atopic dermatitis and acne, hyperproliferative skin diseaseincluding acne, inflammatory bowel disease including Crohn's disease andulcerative colitis, systemic lupus erythematosus, asthma, uveitis,myocarditis, allergy, atherosclerosis, brain inflammation includingAlzheimer's disease and brain inflammatory reaction following traumaticbrain injury, central nervous system disease including spinal cordinjury or cerebral infarction, pathologic angiogenesis including as mayoccur in primary and metastatic tumor growth, rheumatoid arthritis,diabetic retinopathy and atherosclerosis, cancer, chronic pulmonarydisease, acute lung injury, acute respiratory disease syndrome, sepsis,and the like. In addition, S1P₁ receptor agonists are useful fortreating microbial infections, and viral infections or diseases.

One aspect of the present invention relates to methods for treating anS1P₁ receptor-associated disorder in an individual comprisingadministering to the individual in need thereof a therapeuticallyeffective amount of a crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein, a composition as described herein, or apharmaceutical composition as described herein.

One aspect of the present invention relates to methods for treating anS1P₁ receptor-associated disorder in an individual comprisingadministering to the individual in need thereof a therapeuticallyeffective amount of a crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein, a composition as described herein, or apharmaceutical composition as described herein, wherein the disorder isselected from the group consisting of a disease or disorder mediated bylymphocytes, an autoimmune disease or disorder, an inflammatory diseaseor disorder, primary biliary cirrhosis, cancer, psoriasis, psoriaticarthritis, rheumatoid arthritis, Crohn's disease, transplant rejection,multiple sclerosis, systemic lupus erythematosus, inflammatory boweldisease, ulcerative colitis, type I diabetes, and acne.

One aspect of the present invention relates to methods for treating adisorder in an individual comprising administering to the individual inneed thereof a therapeutically effective amount of a crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein, a composition as described herein, or apharmaceutical composition as described herein, wherein the disorder isselected from the group consisting of primary biliary cirrhosis,psoriasis, psoriatic arthritis, rheumatoid arthritis, Crohn's disease,multiple sclerosis, inflammatory bowel disease, systemic lupuserythematosus, and ulcerative colitis.

One aspect of the present invention relates to methods for treating adisease or disorder mediated by lymphocytes in an individual comprisingadministering to the individual in need thereof a therapeuticallyeffective amount of a crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein, a composition as described herein, or apharmaceutical composition as described herein.

One aspect of the present invention relates to methods for treatingprimary biliary cirrhosis in an individual comprising administering tothe individual in need thereof a therapeutically effective amount of acrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein, a composition as described herein, or apharmaceutical composition as described herein.

One aspect of the present invention relates to methods for treatingpsoriasis in an individual comprising administering to the individual inneed thereof a therapeutically effective amount of a crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein, a composition as described herein, or apharmaceutical composition as described herein.

One aspect of the present invention relates to methods for treatingpsoriatic arthritis in an individual comprising administering to theindividual in need thereof a therapeutically effective amount of acrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein, a composition as described herein, or apharmaceutical composition as described herein.

One aspect of the present invention relates to methods for treatingrheumatoid arthritis in an individual comprising administering to theindividual in need thereof a therapeutically effective amount of acrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein, a composition as described herein, or apharmaceutical composition as described herein.

One aspect of the present invention relates to methods for treatingCrohn's disease in an individual comprising administering to theindividual in need thereof a therapeutically effective amount of acrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein, a composition as described herein, or apharmaceutical composition as described herein.

One aspect of the present invention relates to methods for treatingmultiple sclerosis in an individual comprising administering to theindividual in need thereof a therapeutically effective amount of acrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein, a composition as described herein, or apharmaceutical composition as described herein.

One aspect of the present invention relates to methods for treatinginflammatory bowel disease in an individual comprising administering tothe individual in need thereof a therapeutically effective amount of acrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein, a composition as described herein, or apharmaceutical composition as described herein.

One aspect of the present invention relates to methods for treatingsystemic lupus erythematosus in an individual comprising administeringto the individual in need thereof a therapeutically effective amount ofa crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein, a composition as described herein, or apharmaceutical composition as described herein.

One aspect of the present invention relates to methods for treatingulcerative colitis in an individual comprising administering to theindividual in need thereof a therapeutically effective amount of acrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein, a composition as described herein, or apharmaceutical composition as described herein.

One aspect of the present invention relates to methods for treating anautoimmune disease or disorder in an individual comprising administeringto the individual in need thereof a therapeutically effective amount ofa crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein, a composition as described herein, or apharmaceutical composition as described herein.

One aspect of the present invention relates to methods for treating aninflammatory disease or disorder in an individual comprisingadministering to the individual in need thereof a therapeuticallyeffective amount of a crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein, a composition as described herein, or apharmaceutical composition as described herein.

One aspect of the present invention relates to methods for treating amicrobial or viral infection or disease in an individual comprisingadministering to the individual in need thereof a therapeuticallyeffective amount of a crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein, a composition as described herein, or apharmaceutical composition as described herein.

One aspect of the present invention relates to uses of a crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein in the manufacture of a medicament for thetreatment of an S1P₁ receptor-associated disorder.

One aspect of the present invention relates to uses of a crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein in the manufacture of a medicament for thetreatment of an S1P₁ receptor-associated disorder selected from thegroup consisting of a disease or disorder mediated by lymphocytes, anautoimmune disease or disorder, an inflammatory disease or disorder,primary biliary cirrhosis, cancer, psoriasis, psoriatic arthritis,rheumatoid arthritis, Crohn's disease, transplant rejection, multiplesclerosis, systemic lupus erythematosus, inflammatory bowel disease,ulcerative colitis, type I diabetes, and acne.

One aspect of the present invention relates to uses of a crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein in the manufacture of a medicament for thetreatment of an S1P₁ receptor-associated disorder selected from thegroup consisting of primary biliary cirrhosis, psoriasis, psoriaticarthritis, rheumatoid arthritis, Crohn's disease, multiple sclerosis,inflammatory bowel disease, systemic lupus erythematosus, and ulcerativecolitis.

One aspect of the present invention relates to uses of a crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein in the manufacture of a medicament for thetreatment of a disease or disorder mediated by lymphocytes.

One aspect of the present invention relates to uses of a crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein in the manufacture of a medicament for thetreatment of primary biliary cirrhosis.

One aspect of the present invention relates to uses of a crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein in the manufacture of a medicament for thetreatment of psoriasis.

One aspect of the present invention relates to uses a crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein in the manufacture of a medicament for thetreatment of psoriatic arthritis.

One aspect of the present invention relates to uses of a crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein in the manufacture of a medicament for thetreatment of rheumatoid arthritis.

One aspect of the present invention relates to uses of a crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein in the manufacture of a medicament for thetreatment of Crohn's disease.

One aspect of the present invention relates to uses of a crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein in the manufacture of a medicament for thetreatment of multiple sclerosis.

One aspect of the present invention relates to uses of a crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein in the manufacture of a medicament for thetreatment of inflammatory bowel disease.

One aspect of the present invention relates to uses of a crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein in the manufacture of a medicament for thetreatment of systemic lupus erythematosus.

One aspect of the present invention relates to uses of a crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein in the manufacture of a medicament for thetreatment of ulcerative colitis.

One aspect of the present invention relates to uses of a crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein in the manufacture of a medicament for thetreatment of an autoimmune disease or disorder.

One aspect of the present invention relates to uses of a crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein in the manufacture of a medicament for thetreatment of an inflammatory disease or disorder.

One aspect of the present invention relates to uses of a crystallinefree-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein in the manufacture of a medicament for thetreatment of a microbial or viral infection or disease.

One aspect of the present invention relates to uses of a compositioncomprising a crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein and an excipient in the manufacture of amedicament for administration in the treatment of an S1P₁receptor-associated disorder.

One aspect of the present invention relates to uses of a compositioncomprising a crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein and an excipient for preparation a medicamentfor administration in the treatment of an S1P₁ receptor-associateddisorder.

One aspect of the present invention relates to uses of a compositioncomprising a crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein and an excipient for compounding a medicamentfor administration in the treatment of an S1P₁ receptor-associateddisorder.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein for use in a method for the treatment of thehuman or animal body by therapy.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein for use in a method for the treatment of anS1P₁ receptor-associated disorder.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein for use in a method for the treatment of anS1P₁ receptor-associated disorder selected from the group consisting ofa disease or disorder mediated by lymphocytes, an autoimmune disease ordisorder, an inflammatory disease or disorder, primary biliarycirrhosis, cancer, psoriasis, psoriatic arthritis, rheumatoid arthritis,Crohn's disease, transplant rejection, multiple sclerosis, systemiclupus erythematosus, inflammatory bowel disease, ulcerative colitis,type I diabetes, and acne.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein for use in a method for the treatment of anS1P₁ receptor-associated disorder selected from the group consisting ofprimary biliary cirrhosis, psoriasis, psoriatic arthritis, rheumatoidarthritis, Crohn's disease, multiple sclerosis, inflammatory boweldisease, systemic lupus erythematosus, and ulcerative colitis.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein for use in a method for the treatment of adisease or disorder mediated by lymphocytes.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein for use in a method for the treatment ofprimary biliary cirrhosis.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein for use in a method for the treatment ofpsoriasis.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein for use in a method for the treatment ofpsoriatic arthritis.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein for use in a method for the treatment ofrheumatoid arthritis.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein for use in a method for the treatment ofCrohn's disease.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein for use in a method for the treatment ofmultiple sclerosis.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein for use in a method for the treatment ofinflammatory bowel disease.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein for use in a method for the treatment ofsystemic lupus erythematosus.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein for use in a method for the treatment ofulcerative colitis.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein for use in a method for the treatment of anautoimmune disease or disorder.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein for use in a method for the treatment of aninflammatory disease or disorder.

One aspect of the present invention relates to a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described herein for use in a method for the treatment of amicrobial or viral infection or disease.

In some embodiments, the S1P₁ receptor-associated disorder is mediatedby lymphocytes. In some embodiments, the S1P₁ receptor-associateddisorder is primary biliary cirrhosis. In some embodiments, the S1P₁receptor-associated disorder is psoriasis. In some embodiments, the S1P₁receptor-associated disorder is psoriatic arthritis. In someembodiments, the S1P₁ receptor-associated disorder is rheumatoidarthritis. In some embodiments, the S1P₁ receptor-associated disorder isCrohn's disease. In some embodiments, the S1P₁ receptor-associateddisorder is multiple sclerosis. In some embodiments, the S1P₁receptor-associated disorder is inflammatory bowel disease. In someembodiments, the S1P₁ receptor-associated disorder is systemic lupuserythematosus. In some embodiments, the S1P₁ receptor-associateddisorder is ulcerative colitis.

Pharmaceutical Compositions and Compositions

One aspect of the present invention relates to pharmaceuticalcompositions comprising a crystalline free-plate habit of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid as described herein and a pharmaceutical excipient.

One aspect of the present invention relates to pharmaceuticalcompositions comprising a crystalline free-plate habit of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid prepared according to any of the methods described herein and apharmaceutical excipient.

One aspect of the present invention relates to pharmaceuticalcompositions comprising a crystalline free-plate habit of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid (Compound 1) in an amount equivalent to a therapeutically effectiveamount of Compound 1 of the crystalline free-plate habit, wherein thepharmaceutical composition further comprises a diluent, a disintegrant,and a lubricant.

In some embodiments, the diluent comprises a first diluent and a seconddiluent.

In some embodiments, the first diluent comprises mannitol. In someembodiments, the first diluent comprises mannitol 200 SD, USP.

In some embodiments, the second diluent comprises microcrystallinecellulose. In some embodiments, the second diluent comprises Avicel®. Insome embodiments, the second diluent comprises Avicel® PH102, NF.

In some embodiments, the disintegrant is sodium starch glycolate. Insome embodiments, the disintegrant is Explotab®, NF.

In some embodiments, the lubricant is magnesium stearate, NF.

In some embodiments, the pharmaceutical composition has a diluentcontent of about 91% to about 95%.

In some embodiments, the pharmaceutical composition has a first diluentcontent of about 51% to about 55%.

In some embodiments, the pharmaceutical composition has a second diluentcontent of about 37% to about 43%.

In some embodiments, the pharmaceutical composition has a disintegrantcontent of about 3% to about 5%.

In some embodiments, the pharmaceutical composition has a lubricantcontent of about 0.2% to about 0.8%.

In some embodiments, the pharmaceutical composition has:

a crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)-benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid content of 0.69%;

a mannitol content of 54.81%;

a microcrystalline cellulose content of 40%;

a sodium starch glycolate content of 4%; and a magnesium stearatecontent of 0.5%.

In some embodiments, the pharmaceutical composition has:

a crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)-benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid content of 1.381%;

a mannitol content of 54.119%;

a microcrystalline cellulose content of 40%;

a sodium starch glycolate content of 4%; and

a magnesium stearate content of 0.5%.

In some embodiments, the pharmaceutical composition has:

a crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)-benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid content of 2.762%;

a mannitol content of 52.738%;

a microcrystalline cellulose content of 40%;

a sodium starch glycolate content of 4%; and

a magnesium stearate content of 0.5%.

In some embodiments, the pharmaceutical composition has:

a crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)-benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid content of 4.143%;

a mannitol content of 51.357%;

a microcrystalline cellulose content of 40%;

a sodium starch glycolate content of 4%; and

a magnesium stearate content of 0.5%.

One aspect of the present invention relates to compositions comprising acrystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid as described herein or prepared according to any of the methodsdescribed herein.

One aspect of the present invention relates to pharmaceuticalcompositions comprising a crystalline free-plate habit of L-argininesalt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid as described herein or prepared according to any of the methodsdescribed herein, and a pharmaceutical excipient.

In some embodiments, the composition and/or pharmaceutical compositionis suitable for oral, rectal, nasal, topical, buccal, sub-lingual, orvaginal, or in a form suitable for administration by inhalation,insufflation, or by a transdermal patch. In some embodiments, thecomposition and/or pharmaceutical composition is suitable for oraladministration.

Formulations may be prepared by any suitable method, typically byuniformly mixing the active compound(s) with liquids or finely dividedsolid carriers, or both, in the required proportions and then, ifnecessary, forming the resulting mixture into a desired shape.

Conventional excipients, such as binding agents, fillers, acceptablewetting agents, tabletting lubricants and disintegrants can be used intablets and capsules for oral administration. Liquid preparations fororal administration can be in the form of solutions, emulsions, aqueousor oily suspensions and syrups. Alternatively, the oral preparations canbe in the form of dry powder that can be reconstituted with water oranother suitable liquid vehicle before use. Additional additives such assuspending or emulsifying agents, non-aqueous vehicles (including edibleoils), preservatives and flavorings and colorants can be added to theliquid preparations. Parenteral dosage forms can be prepared bydissolving the compound provided herein in a suitable liquid vehicle andfilter sterilizing the solution before filling and sealing anappropriate vial or ampule. These are just a few examples of the manyappropriate methods well known in the art for preparing dosage forms.

The compounds provided herein, such as, the L-arginine salt of Compound1, can be formulated into pharmaceutical compositions using techniqueswell known to those in the art. Suitable pharmaceutically-acceptablecarriers, outside those mentioned herein, are known in the art; forexample, see Remington, The Science and Practice of Pharmacy, 20^(th)Edition, 2000, Lippincott Williams & Wilkins, (Editors: Gennaro et al.).

The term “composition” refers to at least one compound of the inventionin combination with at least one other component. Examples ofcompositions include, but are not limited to, a reference standardcomprising a compound of the present invention (e.g., for use in methoddevelopment, in-process testing, and the like); bulk API (i.e., ActivePharmaceutical Ingredient) of a compound of the present invention (e.g.,for use in formulating a pharmaceutical composition, compounding to forma medicament for use in administrating to a patient, and the like); acombined preparation (i.e., a compound of the present invention incombination with a pharmaceutical/therapeutic agent or agents); abiological sample comprising a compound of the present invention (e.g.,for use in or obtained from a patient, an animal, a pharmacokineticstudy, ADME study, LADME study, and the like); a reaction mixturecomprising a compound of the present invention, such as, a reactionmixture as described in any of the Examples herein; a manufacturingreaction mixture comprising a compound of the present invention incombination with one or more components such as solvents, reactants,side-products, etc.; and the like. It is understood that pharmaceuticalcompositions are a specific subset of compositions. In one embodiment, acomposition can be used in the treatment of a disease or order asdescribed herein.

The compounds provided herein, such as, the L-arginine salt of Compound1, can be “compounded” or be involved in a process of “compounding” toprepare a personalized medication for a patient or a group of patientsfor the treatment of a disease or disorder as described herein.Techniques used in “compounding” are well known to those in the art,such as, the Compounding Pharmacist. In general, “compounding” refers tothe practice of preparing a prescription medication for a specificpatient. Compounding can involve the preparation, mixing, assembling,packaging, and/or labeling of a drug (1) as the result of apractitioner's Prescription Drug Order or initiative based on thepharmacist/patient/prescriber relationship in the course of professionalpractice, or (2) for the purpose of research, teaching, or chemicalanalysis and not for sale or dispensing. Compounding also includes thepreparation of drugs in anticipation of Prescription Drug Orders basedon routine, regularly observed patterns. Compounding services provide avital role in health care as commercially manufactured drugs may not besuited for every person. Due to patient differences, such as, body size,varying drug tolerances, allergies, and/or special requirements (e.g.,dosages that are sugar-free, gluten-free, casein-free, soy-free, and/ordye-free), a patient may require a medication that is not commerciallyavailable with the exact strength needed or may require a medicationthat lacks a certain component found in the commercial drug. Throughcompounding, the pharmacist can adjust the medication to the exactdosage and excipient(s) needed by the patient, for example, a medicationor medicament suitable for oral, rectal, nasal, topical, buccal,sub-lingual, vaginal, parenteral, intramuscular, sub-cutaneous, orintravenous administration, or in a form suitable for administration byinhalation, insufflation, or by a transdermal patch.

While it is possible that, for use in the prophylaxis or treatment, acompound provided herein can, in an alternative use, be administered asa raw or pure chemical, it is preferable however to present the compoundor active ingredient as a pharmaceutical formulation or compositionfurther comprising a pharmaceutically acceptable carrier.

Pharmaceutical formulations include those suitable for oral, rectal,nasal, topical (including buccal and sub-lingual), vaginal or parenteral(including intramuscular, sub-cutaneous and intravenous) administrationor in a form suitable for administration by inhalation, insufflation orby a transdermal patch. Transdermal patches dispense a drug at acontrolled rate by presenting the drug for absorption in an efficientmanner with minimal degradation of the drug. Typically, transdermalpatches comprise an impermeable backing layer, a single pressuresensitive adhesive and a removable protective layer with a releaseliner. One of ordinary skill in the art will understand and appreciatethe techniques appropriate for manufacturing a desired efficacioustransdermal patch based upon the needs of the artisan.

The compounds provided herein, such as, the L-arginine salt of Compound1, together with a conventional adjuvant, carrier, or diluent, can thusbe placed into the form of pharmaceutical formulations and unit dosagesthereof and in such form may be employed as solids, such as tablets orfilled capsules, or liquids such as solutions, suspensions, emulsions,elixirs, gels or capsules filled with the same, all for oral use, in theform of suppositories for rectal administration; or in the form ofsterile injectable solutions for parenteral (including subcutaneous)use. Such pharmaceutical compositions and unit dosage forms thereof cancomprise conventional ingredients in conventional proportions, with orwithout additional active compounds or principles and such unit dosageforms may contain any suitably effective amount of the active ingredientcommensurate with the intended daily dosage range to be employed.

For oral administration, the pharmaceutical composition may be in theform of, for example, a tablet, capsule, suspension, or liquid. Thepharmaceutical composition is preferably made in the form of a dosageunit containing a particular amount of the active ingredient. Examplesof such dosage units are capsules, tablets, powders, granules or asuspension, with conventional additives such as lactose, mannitol, cornstarch or potato starch; with binders such as crystalline cellulose,cellulose derivatives, acacia, corn starch or gelatins; withdisintegrators such as corn starch, potato starch or sodiumcarboxymethyl-cellulose; and with lubricants such as talc or magnesiumstearate. The active ingredient may also be administered by injection asa composition wherein, for example, saline, dextrose or water may beused as a suitable pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition is a solidformulation, such as, a tablet, capsule, suspension for oraladministration, and the like.

The dose when using the compounds provided herein, such as, theL-arginine salt of Compound 1, can vary within wide limits and as iscustomary and is known to the physician, it is to be tailored to theindividual conditions in each individual case. It depends, for example,on the nature and severity of the illness to be treated, on thecondition of the individual, such as a patient, on the compoundemployed, on whether an acute or chronic disease state is treated, orprophylaxis conducted, or on whether further active compounds areadministered in addition to the compounds provided herein, such as, theL-arginine salt of Compound 1. Representative doses include, but are notlimited to, about 0.001 mg to about 5000 mg, about 0.001 mg to about2500 mg, about 0.001 mg to about 1000 mg, about 0.001 mg to about 500mg, about 0.001 mg to about 250 mg, about 0.001 mg to 100 mg, about0.001 mg to about 50 mg and about 0.001 mg to about 25 mg. Multipledoses may be administered during the day, especially when relativelylarge amounts are deemed to be needed, for example 2, 3 or 4 doses.Depending on the individual and as deemed appropriate from thehealthcare provider it may be necessary to deviate upward or downwardfrom the doses described herein.

In some embodiments, the dose is 0.5 mg. In some embodiments, the doseis 1.0 mg. In some embodiments, the dose is 2.0 mg. In some embodiments,the dose is 3.0 mg. In some embodiments, the dose is 5.0 mg.

All dosage amounts disclosed herein are calculated with respect to theactive moiety, i.e., the molecule or ion that gives the intendedpharmacologic or physiologic action.

The amount of active ingredient, or an active salt or derivativethereof, required for use in treatment will vary not only with theparticular salt selected but also with the route of administration, thenature of the condition being treated and the age and condition of theindividual and will ultimately be at the discretion of the attendantphysician or clinician. In general, one skilled in the art understandshow to extrapolate in vivo data obtained in a model system, typically ananimal model, to another, such as a human. In some circumstances, theseextrapolations may merely be based on the weight of the animal model incomparison to another, such as a mammal, preferably a human, however,more often, these extrapolations are not simply based on weights, butrather incorporate a variety of factors. Representative factors includethe type, age, weight, sex, diet and medical condition of theindividual, the severity of the disease, the route of administration,pharmacological considerations such as the activity, efficacy,pharmacokinetic and toxicology profiles of the particular compoundemployed, whether a drug delivery system is utilized, whether an acuteor chronic disease state is being treated or prophylaxis conducted orwhether further active compounds are administered in addition to thecompounds provided herein (e.g. L-arginine salt of Compound 1) such aspart of a drug combination. The dosage regimen for treating a diseasecondition with the compounds and/or compositions provided herein isselected in accordance with a variety of factors as cited above. Thus,the actual dosage regimen employed may vary widely and therefore maydeviate from a preferred dosage regimen and one skilled in the art willrecognize that dosage and dosage regimen outside these typical rangescan be tested and, where appropriate, may be used in the methodsdisclosed herein.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four, or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations. The daily dose can be divided, especially whenrelatively large amounts are administered as deemed appropriate, intoseveral, for example 2, 3, or 4 part administrations. If appropriate,depending on individual behavior, it may be necessary to deviate upwardor downward from the daily dose indicated.

The compounds provided herein, such as, the L-arginine salt of Compound1, can be administered in a wide variety of oral and parenteral dosageforms. It will be obvious to those skilled in the art that the dosageforms may comprise, as the active component, either a compound providedherein or a pharmaceutically acceptable salt, hydrate, or solvate of acompound provided herein.

For preparing pharmaceutical compositions from the compounds providedherein, such as, the L-arginine salt of Compound 1, the selection of asuitable pharmaceutically acceptable carrier can be a solid, liquid, ora mixture of both. Solid form preparations include powders, tablets,pills, capsules, cachets, suppositories and dispersible granules. Asolid carrier can be one or more substances which may also act asdiluents, flavoring agents, solubilizers, lubricants, suspending agents,binders, preservatives, tablet disintegrating agents, or anencapsulating material.

In powders, the carrier is a finely divided solid which is in a mixturewith the finely divided active component.

In tablets, the active component is mixed with the carrier having thenecessary binding capacity in suitable proportions and compacted to thedesired shape and size.

The powders and tablets may contain varying percentage amounts of theactive compound. A representative amount in a powder or tablet maycontain from 0.5 to about 90 percent of the active compound; however, anartisan would know when amounts outside of this range are necessary.Suitable carriers for powders and tablets are magnesium carbonate,magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch,gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, alow melting wax, cocoa butter and the like. The term “preparation”refers to the formulation of the active compound with encapsulatingmaterial as carrier providing a capsule in which the active component,with or without carriers, is surrounded by a carrier, which is thus inassociation with it. Similarly, cachets and lozenges are included.Tablets, powders, capsules, pills, cachets and lozenges can be used assolid forms suitable for oral administration.

For preparing suppositories, a low melting wax, such as an admixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogenous mixture is then poured into convenient sized molds, allowedto cool and thereby to solidify.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or sprays containing inaddition to the active ingredient such carriers as are known in the artto be appropriate.

Liquid form preparations include solutions, suspensions and emulsions,for example, water or water-propylene glycol solutions. For example,parenteral injection liquid preparations can be formulated as solutionsin aqueous polyethylene glycol solution. Injectable preparations, forexample, sterile injectable aqueous or oleaginous suspensions may beformulated according to the known art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a nontoxicparenterally acceptable diluent or solvent, for example, as a solutionin 1,3-butanediol. Among the acceptable vehicles and solvents that maybe employed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The compounds provided herein, such as, the L-arginine salt of Compound1, may thus be formulated for parenteral administration (e.g. byinjection, for example bolus injection or continuous infusion) and maybe presented in unit dose form in ampoules, pre-filled syringes, smallvolume infusion or in multi-dose containers with an added preservative.The pharmaceutical compositions may take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient may be in powder form,obtained by aseptic isolation of sterile solid or by lyophilization fromsolution, for constitution with a suitable vehicle, e.g. sterile,pyrogen-free water, before use.

Aqueous formulations suitable for oral use can be prepared by dissolvingor suspending the active component in water and adding suitablecolorants, flavors, stabilizing and thickening agents, as desired.

Aqueous suspensions suitable for oral use can be made by dispersing thefinely divided active component in water with viscous material, such asnatural or synthetic gums, resins, methylcellulose, sodiumcarboxymethylcellulose, or other well-known suspending agents.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents and thelike.

For topical administration to the epidermis the compounds providedherein, such as, the L-arginine salt of Compound 1, may be formulated asointments, creams or lotions, or as a transdermal patch.

Ointments and creams may, for example, be formulated with an aqueous oroily base with the addition of suitable thickening and/or gellingagents. Lotions may be formulated with an aqueous or oily base and willin general also contain one or more emulsifying agents, stabilizingagents, dispersing agents, suspending agents, thickening agents, orcoloring agents.

Formulations suitable for topical administration in the mouth includelozenges comprising active agent in a flavored base, usually sucrose andacacia or tragacanth; pastilles comprising the active ingredient in aninert base such as gelatin and glycerin or sucrose and acacia; andmouthwashes comprising the active ingredient in a suitable liquidcarrier.

Solutions or suspensions are applied directly to the nasal cavity byconventional means, for example with a dropper, pipette or spray. Theformulations may be provided in single or multi-dose form. In the lattercase of a dropper or pipette, this may be achieved by the patientadministering an appropriate, predetermined volume of the solution orsuspension. In the case of a spray, this may be achieved for example bymeans of a metering atomizing spray pump.

Administration to the respiratory tract may also be achieved by means ofan aerosol formulation in which the active ingredient is provided in apressurized pack with a suitable propellant. If the compounds providedherein, such as, the L-arginine salt of Compound 1, or pharmaceuticalcompositions comprising them, are administered as aerosols, for exampleas nasal aerosols or by inhalation, this can be carried out, forexample, using a spray, a nebulizer, a pump nebulizer, an inhalationapparatus, a metered inhaler or a dry powder inhaler. Pharmaceuticalforms for administration of the compounds provided herein as an aerosolcan be prepared by processes well known to the person skilled in theart. For their preparation, for example, solutions or dispersions of thecompounds provided herein in water, water/alcohol mixtures or suitablesaline solutions can be employed using customary additives, for examplebenzyl alcohol or other suitable preservatives, absorption enhancers forincreasing the bioavailability, solubilizers, dispersants and othersand, if appropriate, customary propellants, for example include carbondioxide, CFCs, such as, dichlorodifluoromethane, trichlorofluoromethane,or dichlorotetrafluoroethane; and the like. The aerosol may convenientlyalso contain a surfactant such as lecithin. The dose of drug may becontrolled by provision of a metered valve.

In formulations intended for administration to the respiratory tract,including intranasal formulations, the compound will generally have asmall particle size for example of the order of 10 microns or less. Sucha particle size may be obtained by means known in the art, for exampleby micronization. When desired, formulations adapted to give sustainedrelease of the active ingredient may be employed.

Alternatively the active ingredients may be provided in the form of adry powder, for example, a powder mix of the compound in a suitablepowder base such as lactose, starch, starch derivatives such ashydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP).Conveniently the powder carrier will form a gel in the nasal cavity. Thepowder composition may be presented in unit dose form for example incapsules or cartridges of, e.g., gelatin, or blister packs from whichthe powder may be administered by means of an inhaler.

The pharmaceutical preparations are preferably in unit dosage forms. Insuch form, the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

Tablets or capsules for oral administration and liquids for intravenousadministration are preferred compositions.

As will be recognized, the steps of the methods provided herein need notbe performed any particular number of times or in any particularsequence. Additional objects, advantages and novel features of theinvention(s) will become apparent to those skilled in the art uponexamination of the following examples thereof, which are intended to beillustrative and not intended to be limiting.

EXAMPLES

The compounds disclosed herein and their syntheses are furtherillustrated by the following examples. The following examples areprovided to further define the invention without, however, limiting theinvention to the particulars of these examples. The compounds describedherein, supra and infra, are named according to the AutoNom version 2.2,CS ChemDraw Ultra Version 9.0.7, or ChemBioDraw Ultra 12.0.2.1076. Incertain instances common names are used and it is understood that thesecommon names would be recognized by those skilled in the art.

Chemistry: Proton nuclear magnetic resonance (¹H NMR) spectra wererecorded on a Bruker Avance III-400 equipped with a 5 mm BBFO probe.Chemical shifts are given in parts per million (ppm) with the residualsolvent signal used as reference. NMR abbreviations are used as follows:s=singlet, d=doublet, dd=doublet of doublets, t=triplet, q=quartet,m=multiplet, bs=broad singlet, sxt=sextet. Microwave irradiations werecarried out using a Smith Synthesizer™ or an Emrys Optimizer™ (Biotage).Thin-layer chromatography (TLC) was performed on silica gel 60 F₂₅₄(Merck), preparatory thin-layer chromatography (prep TLC) was performedon PK6F silica gel 60 Å 1 mm plates (Whatman) and column chromatographywas carried out on a silica gel column using Kieselgel 60, 0.063-0.200mm (Merck). Evaporation was done under reduced pressure on a Büchirotary evaporator. Celite® 545 was used for filtration of palladium.

LCMS spec: HPLC-Agilent 1200; pumps: G1312A; DAD:G1315B; Autosampler:G1367B; Mass spectrometer-Agilent G1956A; ionization source: ESI; DryingGas Flow: 10 L/min; Nebulizer Pressure: 40 psig; Drying Gas Temperature:350° C.; Capillary Voltage: 2500 V) Software: Agilent ChemstationRev.B.04.03.

Example 1: Preparation and Crystallization of L-Arginine Salt of(R)-2-(7-(4-Cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticAcid as Crystalline Spherulite Morphology

A mixture of ethyl2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocylopenta[b]indol-3-yl)acetate(29.0 g, 59.7 mmol, 1.00 equiv.), lipase B (Candida antarctica,immobilized, 1.45 g), and aqueous potassium phosphate buffer (pH7.8±0.2, 1.0 M, 21.75 mL) in acetonitrile (268.5 mL) was stirred undernitrogen at 40° C. for at least 8 hours and until the concentration ofproduct (i.e.,(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid) was ≥40% area by HPLC. Then, while the reaction mixture wasstirred at 20° C., its pH was adjusted to 4 to 6 by addition of 1.0 Maqueous citric acid (26.1 mL). After removal of solids by filtration,washing the reactor then the filter-cake with acetonitrile (3×10 mL),and removal of acetonitrile by vacuum distillation, the product mixturewas partitioned between ethyl acetate (58 mL) and water (58 mL). Theethyl acetate phase was washed with water (2×58 mL) and then with brine(3×58 mL) at least twice and until the pH of the brine wash was 4 to 7and the level of citric acid in the ethyl acetate phase was verified tobe <0.1% by ¹H NMR spectroscopy. Ethyl acetate was replaced with2-propanol (3×87 mL) by vacuum distillation. After complete removal ofethyl acetate was verified by ¹H NMR spectroscopy, the 2-propanol (10mL/g of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid) solution of product was filtered and analyzed by both achiral andchiral HPLC to determine the(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid concentration and to determine the content of the correspondingenantiomer acid (i.e.,(S)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid). The solution was concentrated to an oil and a portion of whichwas used in the preparation of the L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid as described below.

A 2-propanol product mixture containing approximately equal amounts of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid (3.0 g, 6.56 mmol, 1.000 equiv.) and corresponding (S)-ethyl esterwas stirred with 2-propanol (50 mL) at 60° C. After 5 minutes, seedmaterial of L-arginine salt (0.06 g) was added and stirred for 10minutes. Then a preheated 2.27 M aqueous solution of L-arginine (2.89mL, 6.56 mmol, 1.00 equiv.) was added drop-wise over 15 minutes to themixture at 60° C. to form the crystalline L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid. The yellowish suspension was stirred (at ca. 100 rpm) for 60minutes. The mixture was cooled to 25° C.±5° C. at a rate of 0.5° C. perminute. The L-arginine salt was isolated by filtration and washed with2-propanol (3×3 mL) and then ethyl acetate (3×3 mL) until no wet cakeimpurity exceeds 1% area by HPLC. The product was then vacuum dried at40° C.±5° C. for 5 hours, to provide 3.356 g (40.5% yield) crystallineL-arginine salt as spherulites (FIG. 1 shows the spherulites usingscanning electron microscopy (SEM) and FIG. 2 shows the spherulitesusing polarized light microscopy (PLM), with the spherulites exhibitingcharacteristic crosses when visualized under cross polars); HPLC: 99.2%area (achiral), 98.8% w/w (achiral), and 99.5% area (chiral); DSC201.97° C.; particle size: mean 23.58 μm, 11.46 std. dev., 6.7 μmminimum, 47.38 μm maximum length by PLM.

Example 2: Preparation and Crystallization of L-Arginine Salt of(R)-2-(7-(4-Cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticAcid

A mixture of ethyl2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocylopenta[b]indol-3-yl)acetate(21.86 kg, 45.0 mol, 1.000 equiv.), lipase B (Candida antarctica,immobilized, 0.64 kg), and 18.4 kg of aqueous potassium phosphate buffer(pH 7.8±0.2, 1.0 M) in 161.0 kg of acetonitrile was stirred undernitrogen at 40° C.±5° C. for at least 8 hours and until theconcentration of the product (i.e.,(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid) was ≥40% area by HPLC. Then, while the reaction mixture wasstirred at 25° C.±5° C., the pH was adjusted to 4 to 5 by addition of1.0 M aqueous citric acid. After removal of solids by filtration andremoval of acetonitrile by vacuum distillation, the product mixture waspartitioned between ethyl acetate (40.8 kg), water (44.6 kg) and brine(6.7 kg). The ethyl acetate phase was washed with brine (2×54 kg) atleast twice and until the pH of the brine wash was 4 to 6 and the levelof citric acid in the ethyl acetate phase was verified to be <0.1% by ¹HNMR spectroscopy. Ethyl acetate was replaced with 2-propanol (106 kg) byvacuum distillation. After complete removal of ethyl acetate wasverified by ¹H NMR spectroscopy, the 2-propanol solution of product wasdiluted with 53.2 kg of 2-propanol, filtered, and analyzed by bothachiral and chiral HPLC to determine the concentration of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and to determine the corresponding enantiomer acid (i.e.,(S)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid) content.

A 2-propanol product mixture containing approximately equal amounts of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid and the corresponding (S)-ethyl ester was adjusted to 16.7 L/kg ofthe (R)-acid by adding 2-propanol and stirred at 60° C.±5° C. L-argininesalt of Compound 1 seed material (0.18 kg) was added. To the mixture wasadded an aqueous solution of L-arginine (2.27 M, 3.63 kg, 20.8 mol,0.463 equiv. in 9.12 kg of water, preheated to 60° C.±5° C.) tocrystallize the L-arginine salt of Compound 1. The mixture was slowlycooled to 25° C.±5° C. and after crystallization was complete, theL-arginine salt of Compound 1 was isolated by filtration and washed with2-propanol (45 kg) and then ethyl acetate (97.4 kg) until no wet cakeimpurity exceeds 1% area by HPLC. The product was then vacuum dried at40° C.±5° C., sieved, and packaged under nitrogen to provide 12.15 kg(42.7% yield) unmilled L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid. The morphology was found to be different from that described inExample 1. The morphology was characterized as a very fine powder,consisting of small, tightly-layered agglomerates, thin flakes, and veryfine, irregularly-shaped fragments (see FIG. 3A and FIG. 3B). HPLC:99.5% area (achiral); DSC: 204.11° C. (melting onset); Particle size: 14μm (mean length), 1.94 μm minimum and 54.79 μm maximum based on PLM.

Four lots (i.e., Lots J1, J2, J3, and J4) of the L-arginine salt ofCompound 1 totaling 45.82 kg were manufactured from 88.4 kg of ethyl2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocylopenta[b]indol-3-yl)acetatewith Lot J2 being described directly above and Lots J1, J3, and J4 beingprepared by substantially the same procedure as described for Lot J2.Yields for the dry unmilled L-arginine salt based on ethyl ester rangedfrom 39.4 mol % to 43.6 mol % and averaged 42.2 mol %. Total productimpurities based on achiral HPLC ranged from 0.5% area to 1.2% area andaveraged 0.8% area. Levels of the corresponding (S)-acid were less thanthe quantitation limit (0.7% area).

In addition to Lot J2, the morphology of Lot J1 was also analyzed bymicroscopy. The morphology for Lot J1 was characterized as thin lamellar(stacked) flakes, layered into agglomerates that were tightly-layeredand the agglomerates not very porous (see FIG. 4A and FIG. 4B).Differences were observed for the morphologies for Lots J1 and J2.

All four lots were reworked by recrystallization from aqueous 2-propanolas described below in Example 3.

Example 3: Preparation of a Crystalline Plate Habit or Morphology byRecrystallization of L-Arginine Salt of(R)-2-(7-(4-Cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticAcid

A slurry of the L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid (8.20 kg, 12.981 mol) in a mixture of 2-propanol (34.35 kg) and USPwater (7.71 kg) was heated to 75° C. with stirring at 100 rpm undernitrogen atmosphere to furnish a solution which was further heated at78° C. for 0.5 hour. Then, the solution was cooled to 66° C. over 1.5hour and stirred at 61° C. to 66° C. for about 0.75 hour. 2-Propanol(10.05 kg) was added over 1 hour 15 minutes and stirred at 67° C. for 1hour. The slurry was heated to 72° C. to 74° C. to give a solution witha few undissolved particulates and stirred at 81 rpm for 39 minutes atthat temperature. The slurry was cooled to 67° C. over 61 minutes andstirred at 67° C. for 34 minutes and 2-propanol (10.06 kg) was addedover 1 hour 11 minutes to maintain temperature at 65° C. The slurry wasstirred at 66° C. for 1 hour. The slurry was then heated to 70° C. to74° C. for 55 minutes to ensure to keep some undissolved crystals. Theslurry was then cooled to 65° C. over 1 hour and 51 minutes and stirredat 60° C. to 65° C. for an additional 55 minutes and 2-Propanol (10.71kg) was added at 65° C. over 1 hour and 15 minutes. The slurry wasstirred at 66° C. for 1 hour 57 minutes. Then, the slurry was heatedagain to 75° C. over 1 hour, stirred at 75° C. to 78° C. for 40 minutes,and gradually cooled to 67° C. over approximately 1 hour. The slurry wasstirred at about 66° C. to 67° C. for 34 minutes and the slurry wasfurther cooled to 20° C. over 10 hour 48 minutes and continued to stirfor additional 6 hour 10 minutes prior to filtration. The resultantslurry was filtered. The filter cake was rinsed with 19.31 kg of2-propanol. No filtration problems were observed. The wet cake was driedunder vacuum at about 40° C. to provide 7.44 kg (91% yield) of theL-arginine salt of Compound 1 as an off-white solid. The solids weredelumped using a Fitz mill at 1000 rpm using hammer forward position.The milled L-arginine salt (7.36 kg, 89.8% yield) was packaged forstorage. FIG. 5A shows the plates using polarized light microscopy(PLM). DSC: 207.19° C.; PLM: Mean Length: 80 μm, (65 μm std dev), 12 μmminimum and 266 μm maximum. FIG. 5B shows a copy of FIG. 5A that hasbeen modified to add an outline of a substantially intact/completefree-plate with an elongated hexagonal shape.

Four lots of the L-arginine salt (44.95 kg, described in Example 2) wererecrystallized to five L-arginine salt lots totaling 33.19 kg. Lot A3was prepared as described directly above. Lots A1, A2, A4, and A5 wereprepared by substantially the same procedure as described for Lot A3.For these lots, total impurities by achiral HPLC were about 0.1% area,and achiral assays were 100.0% w/w—101.2% w/w and averaged 100.3% w/w.Levels of the corresponding (S)-acid were below the quantitation limitat 0.14% area. The DSC onset temperatures for the five batches are shownbelow in TABLE A1.

TABLE A1 L-Arginine Salt Lot DSC Onset Differential Scanning IsolatedNumber Temperature Calorimetry Morphology A1 ^(a) 208.09° C. FIG. 6Plates A2 ^(a) 207.20° C. FIG. 7 Plates A3 ^(b) 207.19° C. FIG. 8 PlatesA4 ^(a) 207.83° C. FIG. 9 Plates A5 ^(a) 207.90° C. FIG. 10 Plates ^(a)Prepared using a similar process as described in Example 3 ^(b) Preparedas specifically described in Example 3

Example 4: Preparation of a Crystalline Plate Habit or Morphology ofL-Arginine Salt of(R)-2-(7-(4-Cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticAcid from (R/S)-Ethyl2-(7-(4-Cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)acetate

Three methods, referred to as Method 1, Method 2, and Method 3, aredescribed in this example. In each method, the crystalline free-platehabit of the L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid was prepared by enzymatic hydrolysis of the corresponding(R/S)-ethyl ester to the (R)-acid followed by a modified L-argininesalt-forming procedure compared to what was previously disclosed inWO2011/094008. Thereafter, these methods describe isolating theL-arginine salt directly as the crystalline free-plate habit without arecrystallization step.

These methods allow for the direct isolation of the crystallinefree-plate habit of the L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid starting from the corresponding (R/S)-ethyl ester without anydirect isolation of any intermediate.

Example 4.1: Method 1, Preparation of a Crystalline Plate Morphology ofL-Arginine Salt of(R)-2-(7-(4-Cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]-indol-3-yl)aceticAcid

A solution containing 942 mg (6.92 mmol) of potassium phosphatemonobasic dissolved in 7.0 g of water was added to a solution ofpotassium phosphate dibasic containing 12.0 g (68.90 mmol) dissolved69.2 g of water. A check of the buffer solution gave a typical pH of7-8.

To a magnetically stirred two liter two neck round bottom flask wasadded (R/S)-ethyl2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)acetate(100.0 g, 206.0 mmol) and acetonitrile (700.0 g, 886 mL) to form aslurry. The flask was fitted with a reflux condenser and a nitrogen pad.To the slurry was added the potassium phosphate buffer solution followedby 4.0 g of Lipase B, Candida antarctica, immobilized. A check of the pHgave a typical reading of 7-8. The reaction mixture was heated to a 40°C. to 45° C. internal temperature until(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid was present in an amount ≥45% area (but less than 50% area) byHPLC. The reaction mixture was adjusted to a pH 4 to 5 by slow additionof a solution containing 9.0 g (46.84 mmol) of citric acid dissolved in47.0 g of water. The solids were filtered off and washed with 150 g (190mL) of acetonitrile. The filtrates were combined and concentrated to 101g using a rotary evaporator under vacuum (bath at 55° C., pressuredropping to 25 mmHg). The residue was dissolved in 200 g (222 mL) ofethyl acetate. With good stirring, 200 g of water were added followed by39.5 g of saturated sodium chloride brine. After stirring for 5 minutesthe phases were allowed to separate and the organics were washed with asodium chloride solution (3×150 mL, each solution prepared using 100 mLof saturated sodium chloride and 50 mL water). The organics wereconcentrated to 107 g using a rotary evaporator under vacuum (bath at55° C., pressure dropping to 20 mmHg). The residue was dissolved in 80 g(101 mL) of 2-propanol and concentrated to 140 g using a rotaryevaporator under vacuum (bath at 55° C., pressure dropping to 20 mmHg).The residue was diluted with 2-propanol to a weight of 300 g (160 g of2-propanol, 202 mL, was added). The solution was passed through a mediumporosity filter and then transferred to a one liter jacketed reactionvessel. The reaction vessel was equipped with an overhead stirrer,curved stir paddles, reflux condenser, internal temperature probe, andMini Huber recirculating heating/cooling control. An additional 100 g of2-propanol were used in transfer. The product solution, which containeda total of 300 g (379.7 mL) 2-propanol was clear, containing nodetectable solids or cloudiness. To the stirred solution was added 23.4g of water followed by solid L-arginine 17.94 g, (103.0 mmol, 0.5 eq.based on (R/S)-ethyl ester). The reaction mixture was heated to aninternal temperature of 40° C. The reaction mixture changed consistenceafter 5 minutes and then the mixture was heated to an internaltemperature of 76° C. To the slurry was added water drop-wise untilnearly all the solids were in solution (31.5 g). The internaltemperature was adjusted to 80° C. to form a solution. With the internaltemperature at 83° C., 3.3 g of the L-arginine salt were added forseeding. The L-arginine salt dissolved into the solution. The resultingstirred solution was cooled to an internal temperature of 70° C. and2-propanol was added drop-wise until the solution became cloudy (40.3 gadded over 20 minutes). The mixture was heated to an internaltemperature of 75° C.-80° C. and a clear solution formed. To thesolution, an additional amount of 1.2 g of the L-arginine salt was addedfor seeding. To the seeded mixture, 2-propanol was added drop-wise (45 gover 90 minutes). The resulting slurry was held at 80° C. for 10 minutesbefore restarting the drop-wise addition of 2-propanol. Three additions(79.0 g, 78.5 g, and 79.1 g) were done each taking approximately 75minutes with 10 minute holds between. After the last addition, thetemperature controller was programmed to cool the jacket from 85° C. to20° C. over 6 hours and hold.

The stirred mixture was subjected to two cycles of heating to aninternal temperature of 70° C. to 72° C. and cooling to 20 over 4 hours.After the second cycle the mixture was held at 20° C. and a sample waschecked by PLM. The PLM showed only plates (see FIG. 11). The mixturewas heated to an internal temperature of 55° C. to 60° C., held for 1hour and then cool slowly (3 hours) to 30° C. After a 30 minute hold,the mixture was heated to 50° C. to 55° C., held for 1 hour and thencooled slowly (3 hours) to 20° C. and held.

The solids were isolated by filtration using a medium porosity filterpaper and a slight pressure reduction (60 mmHg reduction fromatmospheric). The filter cake was washed (reslurry) with 125 g of2-propanol and followed with a displacement wash with 75 g of2-propanol. The semi-dry cake was washed (reslurry) with 100 g of ethylacetate followed by displacement wash with 100 g of ethyl acetate. Thecake was dried in a vacuum oven (40° C., high house vacuum) to give 65.2g of the L-arginine salt as an off-white solid (plates, see FIG. 12A)with an onset temperature of 205.6° C. by DSC (FIG. 12B).Yield=100×((65.2 g-4.5 g seeds)/65.05 g=93.3% based on (R)-ethyl2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)acetate(i.e., half of the racemic mixture of the starting material ethyl2-(7-(4-cyclopentyl-3-(trifluoromethyl)-benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)acetate).Chiral HPLC analysis showed 99.61% area of Compound 1.

Example 4.2: Method 2, Preparation of a Crystalline Plate Morphology ofL-Arginine Salt of(R)-2-(7-(4-Cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]-indol-3-yl)aceticAcid

A 1.0M buffer solution (pH of 7-8) was prepared by mixing a solution ofpotassium phosphate monobasic (942 mg, 6.92 mmol) in 7.0 g of water witha solution containing potassium phosphate dibasic (12.0 g, 68.90 mmol)in 69.2 g of water.

In a magnetically stirred 2-L three necked round bottom flask fittedwith a reflux condenser and kept under nitrogen, ethyl2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocylopenta[b]indol-3-yl)acetate(100.0 g, 206.0 mmol, 1.000 equiv.) was suspended in acetonitrile (700.0g). To the suspension, the 1.0M potassium phosphate buffer solution wasadded followed by 3.1 g of Lipase B, Candida antarctica. A check of pHgave a typical reading of 7-8. The reaction mixture was then heated inan oil bath at 45° C. until(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid was greater than 44% area by achiral HPLC.

The reaction mixture was cooled to room temperature and the pH of thereaction mixture was adjusted to 4-5 with a slow addition of a solutioncontaining citric acid (9.0 g, 46.84 mmol) dissolved in 47.9 g of water.The solids were filtered off and washed with acetonitrile (150 g). Thefiltrates were combined and concentrated to 121.4 g using a rotaryevaporator under vacuum (bath at 55° C., pressure dropping to 141 mmHg).The residue was dissolved in 200 g of ethyl acetate. With good stirring,200 g of water were added followed by 39.5 g of saturated sodiumchloride brine. After stirring for 5 minutes, the phases were separatedand the organic layer was washed three times sequentially with solutionseach containing 100 mL of saturated sodium chloride brine and 50 mL ofwater. The organics were concentrated to 116.9 g using a rotaryevaporator under vacuum (bath at 55° C., pressure dropping to 139 mmHg).The residue was dissolved in 180 g of 2-propanol. The batch wasconcentrated. The residue was treated again with 180 g of 2-propanol andconcentrated to 121.1 g using a rotary evaporator under vacuum (sameconditions, performed as an ethyl acetate chase). The residue wasdiluted with 2-propanol (113 g) to a weight of 234 g. The solution waspassed through a medium porosity filter and then transferred to a 1-Ljacketed reaction vessel. The reaction vessel was equipped with anoverhead stirrer, curved stir paddles, reflux condenser, internaltemperature probe, and Mini Huber recirculating heating/cooling control.An additional 2-propanol (187.3 g) was used for rinses and transfer.Total amount of 2-propanol was 300.1 g. Material was a solution.

To the stirred solution, L-arginine (17.94 g, 103.0 mmol, 0.500 equiv.based on (R/S)-ethyl ester) was added followed by 53.8 g of water. Thereaction mixture was heated to an internal temperature of 81 C over 1 h15 min to provide a clear solution. At this stage, the water content ofaqueous 2-propanol mixture was 15.2% w/w. With the internal temperatureat 81° C., 2.01 g of seeds (L-arginine salt of Compound 1) were added.The seed crystals did not completely dissolve. The mixture was stirredfor 15 minutes at 80° C.-81° C. To the seeded mixture stirred at 150rpm, 2-propanol was added by a metering pump (329.3 g over 7 h 30minutes). At this stage, the water content of aqueous 2-propanol mixtureis 7.9% w/w. The stir rate was increased to 180 rpm. After the2-propanol addition, the temperature controller was programmed to coolthe jacket from 80° C. to 23° C. over 5 hours and hold at 23° C.

After overnight stirring, a 500 mg sample of the reaction suspension wascentrifuged. The solids were slurried with 0.5 mL of ethyl acetate,centrifuged, and the resultant solids were dried at 40° C. in a vacuumoven at 100 mm Hg. The PXRD overlay of the sample with Lot A5 (plates,see Table A) showed that the sample was the same crystal phase as Lot A5(FIG. 13). The PLM showed predominantly plate morphology with plates upto ˜200 microns by visual observation along with a few aggregates ofplates and plate fragments (FIG. 14).

The reaction suspension was subjected to two cycles of heating to aninternal temperature of 75° C.-78° C., hold for 45 min, and cooling to24° C. over 5 hours. After the first cycle a 500 mg sample of themixture held at 24° C. was centrifuged, and the resultant solids wereslurried in 0.5 mL of 2-propanol. The resultant solids obtained bycentrifuging off 2-propanol was again slurried in 0.5 mL of ethylacetate, centrifuged, dried at 40° C. in a vacuum oven at 100 mm Hg, andchecked for morphology. PLM showed predominantly plate morphology with afew aggregates. DSC showed a bimodal melt. The mixture in the jacketedreactor was held at 24° C. and at a stir rate of 180 rpm.

The solids were isolated by filtration using a medium porosity filterpaper and a slight pressure reduction. The initial deliquoring time was90 minutes. The filter cake was washed with 79 g of 2-propanol (by areslurry method) and with 79 g of 2-propanol (by a displacement method).Total deliquoring time for 2-propanol washes was 45 min. The semi-drycake was washed with 90.2 g of ethyl acetate (by a reslurry method) andwith 90.2 g of ethyl acetate (by a displacement wash). Total deliquoringtime for ethyl acetate washes was 40 min. The cake was dried in a vacuumoven (at 40° C. and 120 mm Hg) to give 60.4 g (46.45% yield based on(R/S)-ethyl ester used for enzymatic resolution and corrected for addedseed amount of L-arginine salt of Compound 1) of the L-arginine salt ofCompound 1 as an off-white solid. Chiral HPLC analysis showed 99.78%area of Compound 1.

Example 4.3: Method 3, Preparation of a Crystalline Plate Morphology ofL-Arginine Salt of(R)-2-(7-(4-Cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]-indol-3-yl)aceticAcid (Lot 06GSp)

A mixture of (R/S)-ethyl2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocylopenta[b]indol-3-yl)acetate(100.0 g, 206.0 mmol, 1.000 equiv.), lipase B (4.0 g, Candidaantarctica, immobilized), and aqueous potassium phosphate buffer (pH7.8±0.2, pH=8.0 for this batch) in acetonitrile (886 mL) was stirredunder nitrogen at 40° C.±5° C. for at least 6 h (24 h for this batch)and until the concentration of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid was ≥40 area % by HPLC (48.4 area % for this batch). Subsequently,while the reaction mixture was stirred at 25° C.±5° C., the pH wasadjusted to 4 to 5 by addition of aqueous citric acid (9.0 g). Afterremoval of solids by filtration and removal of acetonitrile by vacuumdistillation, the product mixture was partitioned between ethyl acetate(222 mL) and water (200 mL). The ethyl acetate phase was washed withbrine at least twice and until the pH was greater than or equal to 4.Ethyl acetate was replaced with isopropanol (twice, 101 mL and 202 mL)by vacuum distillation. After complete removal of ethyl acetate(verified by GC≤0.5 area %, 0.41% for this batch), the isopropanolsolution of product was heated to 60° C.±5° C., then cooled to ≤40° C.,filtered and analyzed by HPLC to determine the concentration of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)aceticacid (free carboxylic acid). Isopropanol was added or removed (vacuumdistilled) to provide a solution of the (R)-free carboxylic acid inisopropanol with a concentration in the range of 11-14 wt %, for thisbatch the concentration was 12.4%.

The isopropanol product solution was treated with the calculated amountof water (23 g) and L-arginine (15.9 g) and heated to 83° C.±2° C. Atthis temperature, water (28.1 g) was added until the solids dissolved.Subsequently, the solution was cooled to 73° C.±2° C. and was treatedwith isopropanol (26.1 g) to obtain a hazy solution. The solution washeated to 75° C.±2° C. and seed crystals of the L-arginine salt of(R)-2-(7-(4-Cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]-indol-3-yl)aceticacid (1.2 g) were added. To the seeded mixture was added isopropanol(280 g) over at least 1 h. Then, the reaction mixture was cooled to 20°C.±2° C. within 6 h.

The heating/cooling cycles were conducted in the following manner. Thestirred product mixture was heated to 71° C.±2° C. and then cooled over4 h to 21° C.±2° C. The mixture was then heated to 60° C.±2° C., stirredat that temperature for 60±10 min before being cooled over 3 h to 31°C.-2° C. After stirring at 31° C.±2° C. for 25±5 min, the stirredmixture was heated to 50° C.±2° C., stirred at that temperature for 45±5min, and then cooled over 3 h to 20° C.±2° C. The heating/cooling cyclescan be represented as 71° C. to 21° C. to 60° C. to 31° C. to 50° C.;temps±2° C. for comparison with the cycles described for other lotsshown in TABLE 2A. Stirring at 20° C.±2° C. was continued for at least 2h before suspension was filtered.

The solids were isolated by filtration (filtration was observed to begood), the filter cake was washed first by reslurrying with isopropanol(100 g), then by displacement with isopropanol (100 g), next byreslurrying with ethyl acetate (100 g), and finally by displacement withethyl acetate (100 g). No single impurity was detected by HPLCanalysis >1.0 area %. The product was vacuum dried at 40° C.±5° C. untilloss on drying (LOD) was passed (≤2%, for a 12 h period), providing 54.9g of the L-arginine salt of(R)-2-(7-(4-Cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]-indol-3-yl)aceticacid as off-white crystalline plates with 100.0 area % by HPLC,L-arginine content of 27.5%, and chiral purity of 99.67%.

Three additional batches were prepared in a manner similar to the aboveprocedure with the following exceptions to the following details shownin TABLE A2.

TABLE A2 Lot 04GSp Lot 05GSp Lot 07GSp Lipase hydrolysis 20 h, 40° C. ±5° C. 22 h, 40° C. ± 5° C. 21 h, 40° C. ± 5° C. conditions pH = 8.0 pH =8.0 pH = 8.0 (R)-Acid = 47.1 area % (R)-Acid = 48.4 area % (R)-Acid =47.9 area % Concentration: 12.4 w/w % 12.4 w/w % 11.7 w/w % (R)-Acid inIPA Heating/Cooling A A B Cycles Product Filtration Good Good Good Final99.8 area % (purity) 99.8 area % (purity) 100.0 area % (purity) L-ArgSalt of Yield: 54.9 g Yield: 56.9 g Yield: 54.7 g (R)-Acid Off-whiteOff-white Off-white crystalline plates crystalline plates crystallineplates L-Arg content: 30.1% L-Arg content: 27.8% L-Arg content: 27.4%Chiral Purity: 99.63% Chiral Purity: 99.52% Chiral Purity: 99.62% A =70° C. to 20° C. to 70° C. to 20° C. to 60° C. to 31° C. to 50° C.;temps are ± 2° C. B = 70° C. to 20° C. to 70° C. to 20° C. to 70° C. to20° C. to 60° C. to 31° C. to 50° C.; temps are ± 2° C.

The DSC onset temperatures for all four batches are shown below in TABLEA3.

TABLE A3 L-Arginine Salt DSC Onset Differential Scanning Isolated LotNo. Lot Number Temperature Calorimetry Morphology 04GSp ^(b) A6 206.55°C. FIG. 22 Plates 05GSp ^(b) A7 206.94° C. FIG. 23 Plates 06GSp ^(a) A8207.04° C. FIG. 24 Plates 07GSp ^(b) A9 207.63° C. FIG. 25 Plates ^(a)Prepared as described in Example 4.3 ^(b) Prepared substantially asdescribed for Lot 06GSp and TABLE A2

Example 5A: Formulations—Evaluation of Spherulites and Plates onFormulation Stability

To evaluate the effect the two morphologies, spherulites and plates, hadon tablet formulation stability of the L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, two batches of core tablets were manufactured using the sameformulation as described in TABLE B and using the same directcompression process described in FIG. 15.

TABLE B ^(a) Batch Component Function % w/w mg/tab Weight (g) L-Arg Saltof API 1.40 1.40 7.00 Compound 1 Mannitol 200 SD, USP Diluent 54.1054.10 270.50 Avicel PH102, NF Diluent 40.00 40.00 200.00 Explotab, NFDisintegrant 4.00 4.00 20.00 Magnesium Stearate, Lubricant 0.50 0.502.50 NF Total Core 100.00 100.00 500.00 ^(a) Batch Size (g) = 500;Tablet Weight (mg) = 100

TABLE C and TABLE D summarize the stability results under theaccelerated storage conditions. After 6 months at the acceleratedconditions, the total impurities (i.e., the sum of the percentages oftwo impurities, referred to a Cmpd A and Cmpd B) were 0.93% for thespherulite formulation and 0.51% for the plate formulation. TheL-arginine salt chemical stability observed in the formulation for thetablets that were manufactured utilizing the plate morphology wasimproved compared to the L-arginine salt chemical stability observed forthe tablets that were manufactured utilizing the spherulite morphology.

Stability data for the spherulite morphology of the L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid after 6 months storage at 40° C./75% RH is shown in TABLE C.

TABLE C 40° C./75% RH Attribute Initial 1 Month 3 Months 6 MonthsStability Data % Label 96.9 97.9 96.8 94.5 Spherulite Cmpd A (%) 0.060.17 0.68 0.61 Morphology Cmpd B (%) 0.04 0.06 0.19 0.32 TotalImpurities 0.10 0.23 0.87 0.93

Stability data for the plate morphology of the L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid after 6 months storage at 40° C./75% RH is shown in TABLE D.

TABLE D 40° C./75% RH Attribute Initial 1 Month 3 Months 6 MonthsStability Data % Label 99.0 98.8 101.5 97.3 Plate Cmpd A (%) 0.06 0.100.60 0.31 Morphology Cmpd B (%) 0.05 0.06 0.17 0.20 Total Impurities0.11 0.16 0.77 0.51

Two batches of core tablets (1 mg and 5 mg, utilizing the L-argininesalt with the plate morphology) were manufactured using the sameformulation as described in Table B (above) and using the same directcompression process described in FIG. 15. Both formulations werepackaged in 40 cc HDPE bottles with desiccant. TABLE E and TABLE F showthe results of a completed 24 month long-term stability study at 25° C.and 60% RH.

TABLE E Stability Data (Plate Morphology) for Tablets (1 mg API) after24 Months 25° C./60% RH 6 9 12 18 24 Attribute Initial Month MonthsMonths Months Months % Label 97.5 96.8 97.2 94.5 96.5 97.2 Cmpd A (%)0.11 0.18 0.20 0.21 0.22 0.25 Cmpd B (%) <QL 0.05 0.05 <QL 0.06 <QLTotal Impurities^(a) 0.11 0.23 0.25 0.21 0.28 0.25 ^(a)Sum ofpercentages of Cmpd A and Cmpd B. QL = Quantitation Limit (0.05%)

TABLE F Stability Data (Plate Morphology) for Tablets (5 mg API) after24 Months 25° C./60% RH 6 9 12 18 24 Attribute Initial Month MonthsMonths Months Months % Label 99.8 98.8 100.2 96.8 97.5 99.3 Cmpd A (%)0.06 0.12 0.13 0.16 0.17 0.19 Cmpd B (%) <QL ND ND <QL <QL <QL TotalImpurities^(a) 0.06 0.12 0.13 0.16 0.17 0.19 ^(a)Sum of percentages ofCmpd A and Cmpd B. QL = Quantitation Limit (0.05%); ND = Not Detected

Example 5B: Formulations for L-Arginine Salt of(R)-2-(7-(4-Cyclopentyl-3-(trifluoromethyl)-benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticAcid

Core tablets were manufactured using the formulation as described inTABLE G and using substantially the same process described in FIG. 15.The amounts of the L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid were calculated according to the percent of parent being 72.42%

TABLE G Tablet Strength 0.5 mg 1 mg 2 mg 3 mg L-Arg Salt of Compound 10.69 1.381 2.762 4.143 Mannitol Pearlito10 100SD 54.81 54.119 52.73851.357 Microcrystalline cellulose-Avicel ® 40 40 40 40 Sodium StarchGlycolate-Explotab ® 4 4 4 4 Magnesium Stearate 0.5 0.5 0.5 0.5 Opadry ®II Blue 4 4 4 4 Total tablet target weight 104 104 104 104

Example 6: Hygroscopicity Comparison Between Plates and Spherulites byDynamic Moisture-Sorption (DMS) Analysis

A dynamic moisture-sorption (DMS) analysis was performed onrepresentative Lot A2 (see, Example 3, TABLE A) of the L-arginine saltof Compound 1 having the plate morphology and representative Lot H2(prepared in a similar manner as described in Example 8, Method 2 StepsG and H in WO2011/094008; and described in TABLE K, Example 8 herein) ofthe L-arginine salt of Compound 1 having the spherulite morphology. TheDMS analysis was performed at 25° C. and the L-arginine salt of Compound1 (plate morphology) was shown to be non-hygroscopic, gaining just 0.2%weight at 90% RH (FIG. 16 and FIG. 17). By comparison the L-argininesalt of Compound 1 (spherulite morphology) gained between 0.5% weight(adsorption cycle) and 0.7% weight (desorption cycle) at 90% RH and 25°C. The plate morphology was observed to have improved hygroscopicitycompared to the spherulite morphology.

Example 7: Powder X-Ray Diffraction Comparison Between Plates andSpherulites

Powder X-ray diffraction (PXRD) showed that Lots A1 to A5 from Example 3(see Table A) were crystalline with matching patterns, indicating thesame crystal phase (see FIG. 18). A PXRD analysis was performed onrepresentative Lot A2 (see, Example 3, TABLE A) of the L-arginine saltof Compound 1 having the plate morphology and representative Lot H2(prepared in a similar manner as described in Example 8, Method 2 StepsG and H in WO2011/094008; and described in TABLE K, Example 8 herein) ofthe L-arginine salt of Compound 1 having the spherulite morphology.Comparing Lot A2 (plate morphology) to Lot H2 (spherulite morphology),it can be seen that both lots share the same crystal phase, however, LotA2 showed better diffraction and lower background noise (i.e., a loweramorphous halo contribution) indicating a higher degree of crystallinityfor the plates compared to the spherulites (FIG. 19).

Certain powder X-ray diffraction peaks for representative Lot A2 of theL-arginine salt of Compound 1 (plates) are shown in TABLE H below.

TABLE H Pos. Height d-spacing Rel. Int. [°2Th.] [cts] [Å] [%] 8.233633.8 10.8159 93.6 14.7 402.2 6.0252 1.1 15.6 364.5 5.6724 1.0 16.44734.0 5.4187 13.2 16.6 931.2 5.3288 2.6 17.2 390.6 5.1667 1.1 19.1927.9 4.6471 2.6 20.1 994.1 4.4250 2.8 20.5 35918.7 4.3373 100.0 22.01056.0 4.0463 2.9 22.2 645.6 4.0046 1.8 22.9 811.2 3.8855 2.3 23.2 671.63.8404 1.9 23.4 1033.2 3.8038 2.9 23.9 1130.9 3.7195 3.2 24.6 3586.13.6159 10.0 25.9 356.9 3.4455 1.0 26.4 363.3 3.3785 1.0 28.8 1899.73.1004 5.3 37.3 1922.3 2.4124 5.4

Certain powder X-ray diffraction peaks for representative Lot H2 of theL-arginine salt of Compound 1 (spherulites) are shown in TABLE I below.

TABLE I Pos. Height d-spacing Rel. Int. [°2Th.] [cts] [Å] [%] 8.2 4469.910.8266 94.9 11.9 1777.7 7.4137 37.7 12.5 1409.5 7.0965 29.9 12.9 1340.96.8507 28.5 13.1 1152.3 6.7346 24.5 13.9 868.3 6.3787 18.4 14.7 844.06.0348 17.9 15.6 750.7 5.6626 15.9 16.6 1356.8 5.3260 28.8 17.1 349.15.1758 7.4 19.0 1954.6 4.6610 41.5 20.0 1752.3 4.4293 37.2 20.5 4712.74.3383 100.0 20.9 2525.3 4.2584 53.6 22.0 1799.9 4.0432 38.2 22.6 1326.43.9363 28.2 23.2 1686.9 3.8367 35.8 24.0 912.9 3.7137 19.4 24.6 953.23.6193 20.2 25.2 386.8 3.5281 8.2 28.9 90.5 3.0857 1.9 30.6 167.1 2.91983.6

Example 8: Evaluation of Preparations of L-Arginine Salt of(R)-2-(7-(4-Cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticAcid

Certain processes for the preparation of the L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid have been previously described, see WO2010/011316 (see Example1.33) and WO2011/094008 (see Example 7, Method 1 Step B; and Example 8,Method 1 and Method 2 Step H).

The L-arginine salt of Compound 1 that was prepared in a similar manneras described in Example 1.33 (WO2010/011316) was observed to have amorphology by PLM as containing aggregates and spherulites along withcrystalline fine particles too small to identify the morphology. Overallthe sample was poorly crystalline and no plates were observed. The sameprocedure was also described in WO2011/094008 (see Example 8, Method 1).In this procedure, the L-arginine salt was prepared starting from thesubstantially pure(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.

The L-arginine salt of Compound 1 prepared from Example 7, Method 1 StepB from WO2011/094008 was observed to have a morphology by PLM ascontaining spherulites with a few fine particles, see FIG. 20. Based onPLM analysis, the particle size for the sample is described in TABLE J.In this example, the L-arginine salt of Compound 1 was prepared in thepresence of the (S)-ethyl ester that remained after the enzymatichydrolysis step.

TABLE J Sample Mean St.Dev Minimum Maximum Example 7, Method 1 Step B ¹18.05 12.18 5.43 58.37 ¹ WO2011/094008

In a similar manner as described in Example 7 Method 1 Step B, theL-arginine salt of Compound 1 was prepared by starting from (R/S)-ethyl2-(7-(4-cyclopentyl-3-(trifluoromethyl)-benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)acetateas described in WO2011/094008, Example 8, Method 2 Steps G and H. Sixlots of the L-arginine salt of Compound 1 were prepared with one lotbeing specifically described in Example 8, Method 2 Steps G and H (i.e.,Lot H1) and five additional lots (i.e., Lots H2 to H6) of the L-argininesalt of Compound 1 being prepared using substantially the sameprocedure. Each lot was observed to have the same crystal phase andspherulite habit with a melting onset temperature ranging from 203.00°C. to 203.97° C. as determined by differential scanning calorimetry(DSC), see TABLE K.

TABLE K Lot Preparation Onset Melting Isolated Number Morphology MethodTemp. (DSC) Morphology H1 Spherulite WO2011/094008 ¹ 203.46° C.Spherulite H2 Spherulite WO2011/094008 ² 203.96° C. Spherulite H3Spherulite WO2011/094008 ² 203.00° C. Spherulite H4 SpheruliteWO2011/094008 ² 203.11° C. Spherulite H5 Spherulite WO2011/094008 ²203.79° C. Spherulite H6 Spherulite WO2011/094008 ² 203.97° C.Spherulite ¹ See Example 8, Method 2 Steps G and H ² Prepared by asimilar process as described for Lot H1

Based on the six lots described in WO2011/094008, four (4) newadditional lots (i.e., J1 to J4) were prepared using substantially thesame procedure (see Example 2). Each of these new lots started with atleast 21.9 Kg of (R/S)-ethyl2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)acetate.Each lot was prepared using the selective enzymatic hydrolysis of(R/S)-ethyl2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)acetateto provide the corresponding(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid (i.e., Compound 1). In the next step and without direct isolationof Compound 1, to the solution (i.e., comprising Compound 1, (S)-ethylester, isopropanol, and water) was added the preheated aqueous solutionof L-arginine to form the L-arginine salt of Compound 1. Afterformation, the crude mixture comprising the L-arginine salt of Compound1 underwent a series of washes and filtration steps as described in FIG.21. Two of the four lots (i.e., Lots J1 and J2) were analyzed bymicroscopy. These two lots were observed to have a different morphology(see Example 2) with respect to each other and each was also observed tobe different from the spherulite morphology or habit observed for thesix lots described in WO2011/094008.

In addition to the formation of different morphologies, it was alsofound that each process used to prepare the four lots of the L-argininesalt of Compound 1 from (R/S)-ethyl2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclo-penta[b]indol-3-yl)acetateresulted in a number of undesired process deviations from that seen forthe six lots described in WO2011/094008. After formation of theL-arginine salt of Compound 1, the resulting product slurry wastransferred to the filter dryer to initiate the series of washes andfiltration steps as described in FIG. 21. Unexpectedly, the filtrationsteps were observed to be excessively long, inefficient, and ultimatelyblinded off the filter. The filtering inefficiencies led to transferringexcessively wet material from the filter dryer to the drying trays thusresulting in further processing delays due to lengthy drying times toachieve specification (i.e., loss on drying ≤2% by weight). Approximatefiltration times are shown in TABLE L.

TABLE L See FIG. 21 Total Total Total Total Total Fil- Batch FiltrationFiltration Filtration Filtration tration Lot Size Time 1 Time 2 Time 3Time 4 Time No. (Kg) (min) (min) (min) (min) (min)¹ J1 22.16 31 82 935378 1426 J2² 21.86 80 228 1371 NR³ 1679 J3 21.80 43 703 6125 564 7435 J422.58 43 570 4643 625 5881 ¹Summation of Total Filtration Times 1 to 4²Prepared as disclosed in Example 2; Lots J1, J3, and J4 were preparedin a similar manner. ³Not Required

Surprisingly, it was found that the novel processes, as describedherein, provided the L-arginine salt of Compound 1 as a free-plate habitinstead of the morphology observed for the previous lots (i.e., the six(6) lots described in WO2011/094008, see TABLE K; and the two lotsanalyzed as described above (Lots J1 and J2), see TABLE L). The platemorphology resulted in a number of advantages, such as, improvedfiltrations, higher degree of crystallinity, improved hygroscopicity,and improved formulation stability.

Example 9: Powder X-Ray Diffraction

Powder X-ray Diffraction (PXRD) data were collected on an X'Pert PRO MPDpowder diffractometer (PANalytical, Inc.) with a Cu source set at 45 kVand 40 mA, Cu(Kα) radiation and an X'Celerator detector. Samples wereadded to the sample holder and smoothed flat with a spatula and weighpaper. With the samples spinning, X-ray diffractograms were obtained bya 12-min scan over the 2-theta range 5-40° 2θ. Diffraction data wereviewed and analyzed with the X'Pert Data Viewer Software, version 1.0aand X'Pert HighScore Software, version 1.0b.

Example 10: Differential Scanning Calorimetry

Differential scanning calorimetry (DSC) study was conducted using a TAInstruments, Q2000 at a heating rate 10° C./min. The instrument wascalibrated for temperature and energy using the melting point andenthalpy of fusion of an indium standard. Thermal events (desolvation,melting, etc.) were evaluated using Universal Analysis 2000 software,version 4.1D, Build 4.1.0.16. Samples were weighed into A1 pans andscans ran from ˜25° C. to ˜270° C. at a rate of 10° C./min.

Example 11: Thermal Gravimetric Analysis

Thermogravimetric analysis (TGA) was conducted using a TA InstrumentsTGA Q500 or Q5000 at a heating rate 10° C./min. The instrument wascalibrated using a standard weight for the balance, and Alumel andNickel standards for the furnace (Curie point measurements). Thermalevents such as weight-loss are calculated using the Universal Analysis2000 software, version 4.1D, Build 4.1.0.16.

Example 12: Dynamic Moisture-Sorption Analysis

DMS analysis was performed using a TA Instruments Q5000 SA (EQ2418).Instrument performance was verified in-house. Samples were added to atared sample holder on the Q5000 SA balance. The sample was dried at 40°C. and then analyzed at 25° C. with an adsorption phase from 30% RH to90% RH and a desorption phase from 90% RH to 10% RH.

Example 13: Microscopy

Microscopy was performed using a Nikon Eclipse E600 POL (EQ0124), with aNikon DS Fil digital camera (EQ0123). Nikon (NIS-Elements BR 3.0)software program was used to collect photomicrographs. Instrumentperformance was verified in-house. Samples were prepared by placing asmall amount of solid on a glass slide, adding a drop of mineral oil toa glass cover slip, placing the oil and cover slip on the sample, andgently pressing down with a gloved finger.

Example 14: BET (Brunauer, Emmett, and Teller) Specific Surface AreaMethod

In general, the specific surface areas for Lots A1-A9 (plates) and H1-H6(spherulites) were determined by physical adsorption of nitrogen gas onthe surface of the sample from each lot using the well-establishedtechnique based on the Brunauer, Emmett, and Teller theory.

The BET surface areas for the samples were measured by MicromeriticsPharmaceutical Servies using a Micromeritics™ TriStar II BET surfacearea analyzer (MicroActive for TriStar II Plus 2.02 Software™). Thesamples were degassed at 25° C. for 960 minutes under vacuum (i.e., 100mm/Hg). The determination of the adsorption of N₂ at 77.3 K was measuredusing a BET surface area eleven point method with relative pressures inthe range of about 0.05 to about 0.3 (P/P₀) for a weighed amount of eachsample, see TABLE M1, TABLE M2, and TABLE N below. The analysis wasperformed per ISO9277.

TABLE M1 Plate Morphology Lot Sample Correlation BET Surface Arena LotNumber (g) Coefficient Area (m²/g) 5015-12-12 A1 0.6163 0.99916 0.75015-12-13 A2 1.5270 0.99945 0.7 5015-12-14 A3 0.4465 0.99922 1.55015-12-15 A4 0.5709 0.99939 1.0 5015-12-16 A5 0.9582 0.99940 0.8

TABLE M2 Plate Morphology Lot Sample Correlation BET Surface Lot No.Number (g) Coefficient Area (m²/g) 04GSp A6 0.4332 0.99921 2.4 05GSp A70.3652 0.99910 1.9 06GSp A8 0.6866 0.99984 3.0 07GSp A9 0.2754 0.999143.1

TABLE N Spherulite Morphology Lot Sample Correlation BET Surface ArenaLot Number (g) Coefficient Area (m²/g) 5015-10-01 H1 0.8891 0.99967 10.05015-10-02 H2 0.6766 0.99967 13.5 5015-10-03 H3 0.7002 0.99966 11.85015-10-04 H4 0.9420 0.99964 12.0 5015-10-05 H5 0.7658 0.99955 10.95015-10-06 H6 0.9662 0.99965 12.3

Those skilled in the art will recognize that various modifications,additions, substitutions, and variations to the illustrative examplesset forth herein can be made without departing from the spirit of theinvention and are, therefore, considered within the scope of theinvention.

1. A method for treating an S1P₁ receptor-associated disorder in anindividual comprising administering to said individual in need thereof atherapeutically effective amount of a crystalline free-plate habit ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid; wherein said crystalline free-plate habit has a BET specificsurface area of about 0.1 m²/g to about 5.0 m²/g.
 2. The methodaccording to claim 1, wherein the crystalline free-plate habit has a BETspecific surface area of about 0.6 m²/g to about 4.0 m²/g.
 3. A methodaccording to claim 1, wherein the crystalline free-plate habit has apowder X-ray diffraction pattern comprising peaks, in terms of 2θ, at8.2°±0.2°, 16.4°±0.2θ, and 20.5°±0.2°.
 4. The method according to claim1, wherein the crystalline free-plate habit has a powder X-raydiffraction pattern comprising peaks, in terms of 2θ, at 8.2°±0.2°,16.4°±0.2°, 20.5°±0.2°, and 24.6°±0.2°.
 5. The method according to claim1, wherein the crystalline free-plate habit has a powder X-raydiffraction pattern comprising peaks, in terms of 2θ, at 8.2°±0.2°,16.4°±0.2°, 20.5°±0.2°, 24.6°±0.2°, 28.8°±0.2°, and 37.3°±0.2°.
 6. Themethod according to claim 1, wherein the crystalline free-plate habithas a differential scanning calorimetry trace comprising an endothermwith an extrapolated onset temperature of 205.0° C. to 208.5° C. at ascan rate of 10° C./minute.
 7. The method according to claim 1, whereinthe crystalline free-plate habit has a differential scanning calorimetrytrace comprising an endotherm with an extrapolated onset temperature of205.5° C. to 208.1° C. at a scan rate of 10° C./minute.
 8. The methodaccording to claim 1, wherein the crystalline free-plate habit has adifferential scanning calorimetry trace conducted at a scan rate of 10°C./minute comprising an endotherm substantially as depicted in any oneof FIGS. 6 to 10 and FIGS. 22 to
 25. 9. The method according to claim 1,wherein the crystalline free-plate habit has a dynamic moisture sorption(DMS) profile with an adsorption phase from 30% RH to 90% RH whereinsaid crystalline free-plate habit gains about 0.3% weight or less at 90%RH.
 10. The method according to claim 1, wherein the crystallinefree-plate habit has: 1) a powder X-ray diffraction pattern comprisingpeaks, in terms of 2θ, at 8.2°±0.2°, 16.4°±0.2°, 20.5°±0.20, and24.6°±0.2°; 2) a differential scanning calorimetry trace comprising anendotherm with an extrapolated onset temperature of 205.5° C. to t208.5° C. at a scan rate of 10° C./minute; 3) a dynamic moisturesorption (DMS) profile with an adsorption phase from 30% RH to 90% RHwherein said crystalline free-plate habit gains about 0.3% weight orless at 90% RH; and/or 4) a BET specific surface area of about 0.6 m²/gto about 4.0 m²/g.
 11. The method according to claim 1, wherein thecrystalline free-plate habit has: 1) a powder X-ray diffraction patterncomprising peaks, in terms of 2θ, at 8.2°±0.2°, 16.4°±0.2°, 20.5°±0.2°,24.6°±0.2°, 28.8°±0.20, and 37.3°±0.2°; 2) a differential scanningcalorimetry trace comprising an endotherm with an extrapolated onsettemperature of 205.5° C. to 208.1° C. at a scan rate of 10° C./minute;3) a dynamic moisture sorption (DMS) profile with an adsorption phasefrom 30% RH to 90% RH wherein said crystalline free-plate habit gainsabout 0.2% weight or less at 90% RH; and/or 4) a BET specific surfacearea of about 0.6 m²/g to about 4.0 m²/g. 12-63. (canceled)
 64. Themethod accordingly to claim 1, wherein said disorder is selected fromthe group consisting of: a disease or disorder mediated by lymphocytes,an autoimmune disease or disorder, an inflammatory disease or disorder,and a microbial or viral infection or disease.
 65. The methodaccordingly to claim 1, wherein said disorder is a disease or disordermediated by lymphocytes.
 66. The method accordingly to claim 1, whereinsaid disorder is an autoimmune disease or disorder.
 67. The methodaccordingly to claim 1, wherein said disorder is an inflammatory diseaseor disorder.
 68. The method accordingly to claim 1, wherein saiddisorder is a microbial or viral infection or disease.
 69. The methodaccordingly to claim 1, wherein said disorder is selected from the groupconsisting of: biliary cirrhosis, primary biliary cirrhosis, autoimmunehepatitis, primary sclerosing cholangitis, primary biliary cholangitis,tissue or solid organ grafts, arthritis, psoriatic arthritis, rheumatoidarthritis, diabetes, type I diabetes and the disorders associatedtherewith, myasthenia gravis, demyelinating disease, multiple sclerosis,ischemia-reperfusion injury, renal ischemia-reperfusion injury, cardiacischemia-reperfusion injury, inflammatory skin disease, psoriasis,atopic dermatitis, acne, hyperproliferative skin disease, inflammatorybowel disease, Crohn's disease, ulcerative colitis, systemic lupuserythematosus, asthma, uveitis, myocarditis, allergy, atherosclerosis,brain inflammation, Alzheimer's disease, traumatic brain injury, spinalcord injury, cerebral infarction, pathologic angiogenesis, primary tumorgrowth, metastatic tumor growth, diabetic retinopathy, cancer, chronicpulmonary disease, acute lung injury, acute respiratory diseasesyndrome, and sepsis.
 70. The method accordingly to claim 1, whereinsaid disorder is selected from the group consisting of: biliarycirrhosis, primary biliary cirrhosis, autoimmune hepatitis, primarysclerosing cholangitis, primary biliary cholangitis, psoriasis, atopicdermatitis, acne, arthritis, psoriatic arthritis, rheumatoid arthritis,inflammatory bowel disease, Crohn's disease, ulcerative colitis,multiple sclerosis, and systemic lupus erythematosus.
 71. The methodaccordingly to claim 1, wherein said disorder is biliary cirrhosis,primary biliary cirrhosis, autoimmune hepatitis, primary sclerosingcholangitis, primary biliary cholangitis, psoriasis, atopic dermatitis,acne, arthritis, psoriatic arthritis, rheumatoid arthritis, inflammatorybowel disease, Crohn's disease, ulcerative colitis, multiple sclerosis,and systemic lupus erythematosus.
 72. The method accordingly to claim 1,wherein said disorder is primary biliary cirrhosis, autoimmunehepatitis, primary sclerosing cholangitis, primary biliary cholangitis,psoriasis, atopic dermatitis, acne, arthritis, psoriatic arthritis,rheumatoid arthritis, inflammatory bowel disease, Crohn's disease,ulcerative colitis, multiple sclerosis, and systemic lupuserythematosus.
 73. The method accordingly to claim 1, wherein saiddisorder is psoriasis.
 74. The method accordingly to claim 1, whereinsaid disorder is atopic dermatitis.
 75. The method accordingly to claim1, wherein said disorder is arthritis.
 76. The method accordingly toclaim 1, wherein said disorder is psoriatic arthritis.
 77. The methodaccordingly to claim 1, wherein said disorder is rheumatoid arthritis.78. The method accordingly to claim 1, wherein said disorder isinflammatory bowel disease.
 79. The method accordingly to claim 1,wherein said disorder is Crohn's disease.
 80. The method accordingly toclaim 1, wherein said disorder is ulcerative colitis.
 81. The methodaccordingly to claim 1, wherein said disorder is multiple sclerosis. 82.The method accordingly to claim 1, wherein said disorder is systemiclupus erythematosus.
 83. A method for preparing a crystalline free-platehabit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, said method comprising the steps of: a) heating a first mixturecomprising L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid, a water-miscible anti-solvent, and water to a first heatingtemperature to form a second mixture; b) cooling said second mixture toa first cooling temperature followed by adding a first additional amountof said water-miscible anti-solvent to said second mixture whilemaintaining said first cooling temperature and thereafter heating to asecond heating temperature to form a suspension; c) cycling Step b)optionally once or twice; and d) cooling said suspension to a secondcooling temperature to form said crystalline free-plate habit ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.
 84. The method according to claim 83, wherein said water-miscibleanti-solvent comprises a solvent selected from the group consisting of:acetonitrile, acetone, tetrahydrofuran, and C₂-C₄ alkanol.
 85. Themethod according to claim 83, wherein said water-miscible anti-solventcomprises 2-propanol.
 89. The method according to claim 83, wherein saidfirst heating temperature is about 60° C. to about 83° C.
 90. The methodaccording to claim 83, wherein after heating to said first heatingtemperature said second mixture is substantially a homogeneous solution.91. The method according to claim 83, wherein Step a) is conducted at astir rate of about 50 rpm to about 250 rpm.
 92. The method according toclaim 83, wherein said first cooling temperature is about 50° C. toabout 70° C.
 93. The method according to claim 83, wherein said cyclingStep b) is conducted once.
 94. The method according to claim 83, whereinsaid cycling Step b) is conducted twice.
 95. The method according toclaim 83, wherein said second heating temperature is at a temperature toretain said suspension.
 96. The method according to claim 83, whereinsaid second cooling temperature is about 50° C. to about 70° C.
 97. Themethod according to claim 83, wherein after cooling to said secondcooling temperature, said method further comprises the step of coolingsaid suspension to a third cooling temperature of about 10° C. to about30° C.
 98. The method according to claim 83, wherein said method furthercomprises the step of isolating said crystalline free-plate habit ofL-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.
 99. The method according to claim 98, wherein said isolatingcomprises filtering said crystalline free-plate habit of L-arginine saltof(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid.
 100. The method according to claim 99, wherein after filtering,said crystalline free-plate habit of L-arginine salt of(R)-2-(7-(4-cyclopentyl-3-(trifluoromethyl)benzyloxy)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl)aceticacid has a BET specific surface area of about 0.1 m²/g to about 5.0m²/g.